Assay methods for group transfer reactions

ABSTRACT

The present invention relates to methods for detecting, quantifying and high throughput screening of donor-products and the catalytic activities generating the donor-products in group-transfer reactions. The invention further provides immunoassays, antibodies and kits that may be used to practice the methods of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/443,746 filed Jan. 30, 2003, which is incorporated by reference herein its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with United States government support awarded bythe National Institutes of Health under grant numbers: GM59542, GM69258and CA110535. The United States government has certain rights in thisinvention.

TECHNICAL FIELD

The present invention relates to group transfer reaction methodologies.The invention provides methods for the detection and quantification ofdonor-products and the catalytic activities generating thedonor-products in group transfer reactions. The invention also providesmethods for high throughput screening of acceptor substrates,inhibitors, or activators of enzymes catalyzing group transferreactions. The invention further provides immunoassays, antibodies andrelated kits for practicing the invention.

BACKGROUND OF THE INVENTION

There are many important biological reactions where the substrates aremodified by chemical groups that are donated by other substrates, knownas activated donor molecules. These biological reactions are broadlyrecognized as “group transfer reactions” and have the general reaction:donor-X+acceptor→donor-product+acceptor-X.Typically, donor-X, the activated donor molecule, is a nucleotideattached to a covalent adduct. The donor-X is activated by formation ofa phosphoester bond in the nucleotide donor. Also the acceptorsubstrates can include small molecules or macromolecules such asproteins or nucleic acids. Products of this reaction are the modifiedacceptor, acceptor-X and the donor-product molecule.

There are many enzymes that catalyze group transfer reactions such asfor example kinases, which use ATP to donate a phosphate group;sulfotransferases (SULTs), which use phosphoadenosine-phosphosulfate(PAPS) to donate a sulfonate group; UDP-glucuronosyltransferases (UGTs),which use UDP-glucuronic acid to transfer a glucuronic acid group;methyltransferases, which use s-adenosyltransferase to donate a methylgroup; acetyl transferase, which use acetyl coenzymeA to donate anacetyl group; and ADP-ribosyltransferases, which use nicotinamideadenine dinucleotide (NAD) to donate an ADP-ribose group. Thus, many ofthe enzymes catalyzing group transfer reactions are of interest topharmaceutical companies.

Automated high throughput screening (HTS) assays are the paradigm foridentifying interactions of potential drug molecules with proteins in adrug discovery setting, and this format requires simple, robustmolecular assays, preferably with a fluorescent or chemiluminescentreadout. The most suitable format for HTS is a homogenous assay, (e.g.,“single addition” or “mix and read” assay), which does not require anymanipulation after the reaction is initiated and the assay signal can bemonitored continuously. Despite their importance from a drug discoveryperspective, the incorporation of group transfer enzymes intopharmaceutical HTS programs is being slowed or prevented for a number ofreasons: a) homogenous assay methods are not currently available, b) thedetection methods used place serious limits on the utility of the assay,and c) the non-generic nature of the assay requires the development ofmany specific detection reagents to test diverse acceptor substrates.

The approach currently used to identify sulfotransferase substrates orinhibitors requires the use of radioactivity and involves cumbersomepost-reaction separation steps, such as precipitation or chromatography.For instance, ³⁵S-PAPS is used in a sulfotransferase reaction and thelabeled product is quantified by scintillation counting after selectiveprecipitation of unreacted ³⁵S-PAPS (Foldes, A. and Meek, J. L., BiochimBiophys Acta, 1973, 327:365-74). This approach is not desirable in ahigh throughput screening (HTS) format because of the high radiationdisposal costs and because the incorporation of separation stepscomplicates the automation process. Other SULT assays have beendeveloped using calorimetric and fluorescent means, but they aredependent on the use of a specific acceptor substrate for detection, sotheir use is limited to a single SULT isoform, and they cannot be usedto screen for diverse substrates (Burkart, M. D. and Wong, C. H., AnalBiochem, 1999, 274:131-7; Frame, L. T., et al., Drug Metab Dispos, 2000,28:1063-8). As a result, SULT interaction studies are currently notincluded during the preclinical development of drugs. Also UGTs arecurrently assayed using radiolabeled donor molecules and requirepost-reaction separation steps such as thin layer chromatography (TLC)or high pressure liquid chromatography (HPLC) which seriously hamperspreclinical HTS programs (Ethell, B. T., et al., Anal Biochem, 1998,255:142-7). Likewise, traditionally, kinases have been assayed by filtercapture or precipitation of radiolabeled polypeptide substrates producedusing ³²P-ATP or ³³P-ATP as donor. However, since this method requires aseparation step such as filtering or centrifugation, it cannot be easilyadapted to an automated HTS format. Surface proximity assays (SPA) allowradioassays in a multiwell format with no separation (Mallari, R., etal., J Biomol Screen, 2003, 8:198-204) but their use by pharmaceuticalcompanies is declining because of the disposal and regulatory costs ofhandling radioisotopes.

Because of the high level of interest in developing kinase inhibitordrugs, there has been a great deal of effort, scientists to developimproved assay methods for this enzyme family. Homogenous assay methodshave been developed, in which highly specific reagents are used todetect the reaction products in the presence of the other components ofthe reaction using a light-based readout, such as fluorescence orchemiluminescence. The most common homogenous approach used for kinaseassays is immunodetection of phosphopeptide products exhibitingdifferent fluorescence properties (Zaman, G. J., et al., Comb Chem HighThroughput Screen, 2003, 6:313-20). In this method, phosphorylation ofsubstrate peptide leads to displacement of a fluorescent phosphopeptidetracer from anti-phosphopeptide and causes a change in its fluorescenceproperties. This basic approach has been adapted to several differentreadout modes used for competitive immunoassays including Fluorescencepolarization (FP) (Parker, G. J., et al., J Biomol Screen, 2000,5:77-88); time resolved fluorescence (Xu, K., et al., J Biochem MolBiol, 2003, 36:421-5); fluorescence lifetime discrimination (Fowler, A.,et al., Anal Biochem, 2002, 308:223-31); and chemiluminescence (Eglen,R. M. and Singh, R., Comb Chem High Throughput Screen, 2003, 6:381-7).

The shortcoming with this approach is the requirement forphosphopeptide-specific antibodies. Though generic phosphotyrosineantibodies are common, phosphoserine and phosphothreonine antibodies arenotoriously difficult to produce and only recognize phospho-serine or-threonine in the context of specific flanking amino acids (Eglen andSingh, 2003). There are over 400 kinases in humans and their specificityfor phosphorylation sites vary widely. Thus, different antibodies areneeded for assaying diverse kinases or profiling acceptor substrates.This greatly complicates the incorporation of new kinases into HTS,especially if their substrate specificity is not well defined. It alsocreates analysis problems in comparing data among kinases with differentsubstrate selectivities, because the output of the assay depends on theparticular antibody (Ab)-phosphopeptide pair used. Although efforts todevelop generic phospho-serine antibodies and identify more generickinase substrates continue (Sills, M. A., et al., J Biomol Screen, 2002,7:191-214), research in this direction has not been very successful todate.

A number of alternative approaches have been developed to circumvent theproblem of context-specific Ab-phosphopeptide interactions, includinguse of metal complexes to bind phosphopeptides (Scott, J. E. andCarpenter, J. W., Anal Biochem, 2003, 316:82-91) and the use of modifiedATP analogs that allow covalent tagging of phosphopeptide products(Allison Miller-Wing, E. G., Barbara Armstrong, Lindsey Yeats, RamBhatt, Frank Gonzales, and Steven Gessert., SBS 9th Annual Conferenceand Exhibition, Portland, Oreg., 2003). Chemical phosphate bindingreagents suffer from background binding to nucleotide phosphates,requiring the use of very low, non-physiological levels of ATP andlimiting assay flexibility. Modified nucleotides do not provide ageneric format because the ability to use the ATP analogs as donorsvaries among kinases, requiring the development of a number of differentanalogs. Also competition of inhibitors with the modified nucleotides atthe kinase ATP binding site—the most frequent site for kinase inhibitorbinding—does not reflect the physiological situation. Differences inprotease sensitivity caused by peptide phosphorylation have also beenexploited in developing fluorescence based kinase assays (Kupcho, K., etal., Anal Biochem, 2003, 317:210-7), but these assays are not trulyhomogenous; i.e. they require the post-reaction addition of developingprotease reagents. In addition, the applicability of this method islimited to peptides where kinase and protease specificity overlap.

There are also a few methods that are dependent on interaction ofreaction products with specific multiwell plate chemistries, but theseare not truly homogenous in that they require post reaction reagentadditions and/or processing. Also the requirement for specializedinstrumentation for processing and/or detection does not fit with theopen architecture desired by most pharmaceutical HTS platforms.

Furthermore, microfluidics-based kinase assays that rely onelectrophoretic separation of reaction products have been developed(Xue, Q., et al., Electrophoresis, 2001, 22:4000-7). In these assays,phosphorylated peptide products are electrophoretically separated fromthe non-phosphorylated acceptor substrates, thus eliminating the needfor specialized detection reagents. However, in practice, the kinaseassays are often run in multiwell plates and then the products aretransferred to microfluidic devices for separation—a cumbersome processfor an HTS format.

In summary, the non-generic nature of the current group transfer assaysis resulting in significant expense and delays for drug discoverybecause of the need to develop assays for individual enzymes or smallsubgroups within a family. Also, because many of the current assays arebased on modification and detection of specific tagged acceptors, thereis limited ability for testing different acceptor substrates. Often thetagged acceptor substrates used are different from the substrates thatare phosphorylated in vivo, thus the physiological relevance of theassay is questionable. In addition, a major concern in thepharmaceutical industry is that because of the non-generic nature of thecurrent assays, investigators are sometimes forced to use differentmethods for different kinases. However, studies have shown that thereare significant differences in the pharmaceutical targets identifiedusing different assays methods (Sills, M. A., et al., J Biomol Screen,2002, 7:191-214), which is a significant problem for profiling inhibitorselectivity across several kinases. These shortcomings of the existingHTS assay methods for group transfer reactions are hampering the rapidanalysis of important enzyme families in pharmaceutical drug discoveryprograms.

Other existing approaches for assaying group transfer reactions havebeen to enable screening of diverse chemicals as substrates for grouptransfer reactions by detecting the donor molecule product, because itis the same regardless of the acceptor being modified. Detection of thedonor product has been thought to provide the basis of a generic assaymethod, ADP is always a product of a kinase reaction andphosphoadenosine-phosphate (PAP) is always the product of asulfotransferase reaction. Detection of these products, however, iscomplicated because the cleaved mono- and di-nucleotides cannot bedifferentiated from the activated donor molecules based on absorbance orfluorescence properties because for example ADP has the samefluorescence and absorbance properties as ATP. Separation of the donorproduct from the donor can be effected using chromatographic methods,such as thin layer chromatography or high pressure liquidchromatography, but incorporation of these methods into an HTS format iscumbersome.

To circumvent this difficulty, detection of the donor product has beenachieved by using additional enzymes to generate a detectable productfrom the primary reaction product—the cleaved mono- or di-nucleotide;this is known as an enzyme coupled reaction. For instance, enzymes andother small molecules can be used for ADP-dependent generation of NADPH,which is detected by absorbance or fluorescence at 340 nm (Walters, W.P. and Namchuk, M., Nat Rev Drug Discov, 2003, 2:259-66). An enzymecoupled reaction has also been developed for UGTs, another type of grouptransfer enzyme (Mulder, G. J. and van Doorn, A. B., Biochem J, 1975,151:131-40). However, the optical interference of drug compounds withabsorbance assays, especially in the ultra violet, is a widelyrecognized problem with this approach. Another shortcoming of thisapproach is that all of the enzymes used to couple the detection aresubject to potential inhibition from the chemicals being screened.

Another generic approach is to monitor ATP consumption using Luciferaseas a reporter to detect protein kinase activity. An example of thismethod was disclosed by Crouch et al., in U.S. Pat. No. 6,599,711. Theirmethod entailed determining the activity of a protein kinase to betested by adding a substrate capable of being phosphorylated by theprotein kinase to a solution having ATP and a protein kinase to betested, and another solution having ATP in the absence of the kinase tobe tested. The concentration or the rate of time change of ATP and/orADP was then measured using bioluminescence. However this assay is notoptimal because it relies on small decreases in a high initial signal.The need to keep ATP concentrations low to minimize background resultsin nonlinear reaction kinetics if assay conditions are not carefullycontrolled. In a related method, competition binding assays usingfluorescent ATP analogs have also been developed, but these do not givea measure of enzyme catalytic activity, thus are of limited utility.

The use of enzymes that catalyze group transfer reactions into HTSassays has been hampered by the lack of universal, homogenous assaymethods that are not subject to interference from molecules inpharmaceutical drug libraries. Accordingly, it would generally bedesirable to provide universal methods for assaying enzymes involved ingroup transfer reactions that are well suited for HTS drug discovery.

BRIEF SUMMARY OF THE INVENTION

To enable facile incorporation of important group transfer enzymes intoHTS assays, applicants have developed assays based on homogenousimmunodetection of the donor product. The general equation for the grouptransfer reaction is: donor-X+acceptor→donor-product+acceptor-X, whereinthe donor-product is detected by the general detection reaction: firstcomplex+donor-product→second complex+displaced detectable tag. Becausethe donor product is the same for all enzymes that catalyze a given typeof group transfer reaction, the same detection reagents can be used forall the members within a family of group transfer enzymes and with anyacceptor substrate. The assay products can be detected using homogenousfluorescence or chemiluminescence methods which are not subject tosignificant interference or background signal from molecules inpharmaceutical drug libraries.

Thus, the present invention is summarized as methods and componentsthereof for detecting the activity of and screening acceptor substrates,inhibitors, or activators of enzymes catalyzing group transfer reactionsto facilitate the development of more selective and therapeutic drugs.This is accomplished through a highly selective antibody used to bindthe donor product of the group transfer reaction. Antibody-antigeninteractions can be detected in a number of ways that have already beendescribed by others. The detection mode applicants have used to put themethod into practice is a competitive fluorescence polarizationimmunoassay (FPIA), because it is well suited for pharmaceutical HTSassays. With this detection mode, enzymatically generated donor productdisplaces a fluorescent derivative of the donor product, called atracer, from an antibody resulting in a decrease in tracer fluorescencepolarization. The key reagents for the assay are an antibody that bindswith high selectivity to the donor product, and not to the uncleaveddonor molecule, and a tracer—a fluorescent derivative of the donorproduct that retains its structure sufficiently to bind the antibody.The invention provides a novel assay for detecting and quantifyingactivity for enzymes that catalyze group transfer reactions usingdiverse substrates. The invention also provides a method of screeningfor substrates, inhibitors, or activators of the group transferreactions.

In one aspect, the invention provides a method of detecting adonor-product of a group transfer reaction, the method includingreacting an activated form of a donor with an acceptor in the presenceof a catalytically active enzyme; forming the donor-product and anacceptor-X; contacting the donor-product with a first complex comprisinga detectable tag capable of producing an observable; competitivelydisplacing the detectable tag of the first complex by the donor productto generate a second complex and a displaced detectable tag; anddetecting a change in the observable produced by the detectable tag inthe first complex and the displaced detectable tag.

In another aspect, the invention provides an antibody produced against adonor product of a group transfer reaction, wherein the antibody has theability to preferentially distinguish between a donor-product and adonor in the presence of a high donor concentration.

In yet another aspect, the invention provides a homogeneous competitivebinding assay for a donor product of a group transfer reaction, theassay includes combining the donor-product with a tracer and amacromolecule to provide a mixture, the macromolecule being specific forthe donor product, the tracer comprising the donor-product conjugated toa fluorophore, the tracer being able to bind to the macromolecule toproduce a detectable change in fluorescence polarization; measuring thefluorescence polarization of the mixture to obtain a measuredfluorescence polarization; and comparing the measured fluorescencepolarization with a characterized fluorescence polarization value, thecharacterized fluorescence polarization value corresponding to a knowndonor-product concentration.

In yet another aspect, the invention provides assay kits forcharacterizing a donor-product from a group transfer reaction, the assaykit including a macromolecule and a tracer, each in an amount suitablefor at least one homogeneous fluorescence polarization assay fordonor-product, wherein the macromolecule is a an antibody or aninactivated enzyme.

Other advantages and a fuller appreciation of specific adaptations,compositional variations, and physical attributes will be gained upon anexamination of the following detailed description of the variousembodiments, taken in conjunction with the appended claims.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 illustrates a fluorescence polarization immunoassay (FPIA)reaction for the SULT reaction product, PAP.

FIG. 2 illustrates use of FPIA to detect and quantify UDP formation, thedonor product of the UGT reaction.

FIG. 3 illustrates use of FPIA to detect and quantify ADP formation, thedonor product of the kinase reaction.

FIG. 4 describes a strategy for iterative co-development of reagents forSULT1E1 FPIA: anti-PAP antibody and fluorescently labeled PAP detectabletag.

FIGS. 5A-B show titration or competitive displacement curves for uridinenucleotides using a polyclonal antibody raised against UDP/UTP and acommercially available tracer molecule (ALEXA™-UTP (a fluorescent dyesynthesized through sulfonation of amino-coumarin or rhodamine andproduced by Invitrogen, Carlsbad, Calif.)).

FIG. 6 shows the synthesis of PAP antigens.

FIGS. 7A-C show components of PAP tracer synthesis.

FIGS. 8A-C show representative final tracer structures.

FIG. 9 shows binding isotherms for anti-PAP antibodies andPAP-fluorescein tracers.

FIG. 10 shows competitive displacement of two different tracers from Ab3642 by PAP and PAPS.

FIGS. 11A-B show the competition curves with two Anti-PAPantibody/Tracer combinations.

FIG. 12 shows the effect of PAPS concentration on detection ofenzymatically generated PAP.

FIGS. 13A-F show a continuous FPIA-based detection of SULT activity withdiverse substrates.

FIG. 14 shows a comparison of SULT1E1 acceptor substrate profilesdetermined using the FPIA-based assay and the ³⁵S-PAPS radioassay.

FIG. 15 shows inhibition of SULT1E1 by 2,6 Dichloro-4-nitrophenol (DCNP)measured with the FPIA-based assay.

Before an embodiment of the invention is explained in detail, it is tobe understood that the invention is not limited in its application tothe details set forth in the following description. The invention iscapable of other embodiments and of being practiced or being carried outin a variety of ways. Also, it is to be understood that the phraseologyand terminology used herein is for the purpose of description and shouldnot be regarded as limiting in any way.

DETAILED DESCRIPTION OF THE INVENTION

The present invention broadly relates to novel assay methods fordetecting and quantifying the donor-product or the catalytic activitiesgenerating the donor-product from group transfer enzymes using diversesubstrates. The invention also provides the antibodies specific to thedonor product and assay kits for practicing the invention in ahigh-throughput screening format. The general equation for the grouptransfer reaction includes a donor-X+acceptor→donor-product+acceptor-X,wherein the donor-product is detected by the general detection reaction:first complex+donor-product→second complex+displaced detectable tag.Thus, in accordance with the invention, a highly selective antibody isused to bind the donor product of the group transfer reaction, and thisbinding event is detected, using an immunoassay, such as for example acompetitive binding FPIA which is well suited for pharmaceutical HTSassays. By using this detection mode, enzymatically generated donorproduct displaces a tracer, from an antibody resulting in a decrease intracer fluorescence polarization. The key reagents for the assay areantibody that binds with high selectivity to the donor product, and notto the uncleaved donor molecule, and a tracer—a fluorescent derivativeof the donor product that retains its structure sufficiently to bind theantibody.

In particular the invention provides a universal assay method in that asingle set of detection reagents can be used for all of the enzymes in agiven family of group transfer enzymes and all acceptor substrates forthat family. Because of its universal nature, the invention willaccelerate the incorporation of SULTs, protein kinases and other grouptransfer enzymes into HTS screening. For example, there are eleven knownSULT isoforms, using the method of the present invention, all eleven ofthe SULT isoforms may be screened for their ability to sulfate diversecompounds in the same experiment using the same detection reagents,protocol and instrumentation. This is an important capability becauseenzymes that catalyze xenobiotic conjugation (e.g., SULTs and UGTs) havevery broad acceptor substrate specificity. With respect to kinases,there is even more diversity within the enzyme family. There are over400 protein kinases in humans, and there is great diversity in theiracceptors substrate specificity such that either physiological proteinacceptor substrates as well as short peptides may be used. As such, anumber of protein kinases may be used in the assay of the inventionusing their diverse acceptor substrates and screened for inhibitorsusing the same detection reagents, protocol and instrumentation. Forthese reasons and others provided below, the FPIA based donor productdetection assays of the invention for group transfer reactions such asSULT, UGT and kinases, among others are very well suited for automatedHTS applications.

Accordingly, the present invention will now be described in detail withrespect to such endeavors; however, those skilled in the art willappreciate that such a description of the invention is meant to beexemplary only and should not be viewed as being limiting on the fullscope thereof.

Definitions Certain Terms Used Herein are Intended to have the FollowingGeneral Definitions

The term “group transfer reaction” as used herein refers to the generalreaction:donor-X+acceptor donor→product+acceptor-X.

Representative group transfer reactions are shown as follows:

-   Kinase reaction: ATP+acceptor→ADP+acceptor-PO₄;-   UGT reaction: UDP-glucuronic acid+acceptor→UDP+acceptor-glucuronic    acid;-   SULT reaction: acceptor-XH+3′-phosphoadenosine    5′-phosphosulfate→acceptor-SO₄+3′-phosphoadenosine 5′-phosphate+H⁺;-   Methyltransferase reaction:    s-adenosylmethionine+acceptor→acceptor-CH₃+s-adenosylhomocysteine;    and-   Acetyl transferase reaction: acetyl    CoenzymeA+acceptor→acceptor-COCH₃+CoenzymeA.

Group transfer reactions in-part such as sulfation by SULTs,phosphorylation by kinases and UDP-glucuronidation by UGTs are suitablyapplicable to the methods of the invention because one can isolateantibody/detectable tag pairs for the donor products, which are PAP, ADPand UDP, respectively. Also group transfer reactions are generallyinvolved in a number of biological processes, such as hormonebiosynthesis and function; enzyme regulation and function; andxenobiotic metabolism.

The term “universal assay” and “generic assay” are used interchangeablyto refer to a method whereby all members of the group transfer reactionenzyme family and all of their acceptor substrates can be detected withthe same assay reagents.

The term “covalent adduct” refers to the moiety that is transferred fromthe donor molecule to the acceptor in a group transfer reaction;sulfonate, phosphate, and glucuronic acid respectively for SULTs,kinases, and UGTs.

The term “donor-product” as used herein refers to the product of a grouptransfer reaction that is the fragment of the donor molecule that isgenerated when the covalent adduct is transferred to acceptor. Often itis a nucleotide (naturally occurring or synthetic) such as a PAP, UDP orADP; or a non-nucleotide such as a s-adenosylhomocysteine, nicotinamideor a CoenzymeA. The donor-product is detected by a general reactionincluding a first complex+donor-product→second complex+displaceddetectable tag.

The term “tracer” as used herein refers to a displaced detectablederivative or tag of a donor product that retains its structuresufficiently to bind to a specific antibody.

The term “donor” as used herein refers to a substrate for an enzymecatalyzing a group transfer reaction that carries the activated covalentadduct. Examples of suitable donors include not only nucleotides, butalso s-adenosyl methionine and acetyl-CoA, among others.

The term “donor-X ” is another term for donor molecule in which Xrepresents the covalent adduct.

The term “acceptor” as used herein refers to a substrate for an enzymecatalyzing a group transfer reaction to which the covalent adductbecomes covalently attached, wherein the substrate is a polypeptide, aprotein, a nucleic acid, a carbohydrate, a lipid or a small moleculesubstrate such as a steroid or an amino acid.

The term “acceptor-X” as used herein refers to a reaction product inwhich X is the covalently bound covalent adduct; wherein the covalentadduct includes at least one of a phosphate, a sulfate, a carbohydrate,a naturally occurring amino acid, a synthetically derived amino acid, amethyl, an acetyl, or a glutathione moiety, and a combination thereof.The covalent adduct is capable of altering either the function, thestability, or both the function and the stability of the acceptorsubstrate.

The term “catalytically active enzyme” as used herein refers to at leastone of a sulfotransferase, a kinase, a UDP-glucuronosyltransferase, amethyl transferase, an acetyl transferase, a glutathione transferase, ora ADP-ribosyltransferase.

The term “catalytic activity” as used herein refers to a chemicalcatalytic activity, an enzymatic activity, or a combination thereof.

The term “first complex” as used herein refers to a complex having amacromolecule (i.e., an antibody or an inactivated enzyme) and adetectable tag.

The term “second complex ” as used herein refers to a macromolecule andthe donor product wherein the detectable tag is competitively displacedby the donor-product.

The term “observable” as used herein refers to detectable change influorescence, fluorescence intensity, fluorescence lifetime,fluorescence polarization, fluorescence resonance energy transfer (FRET)or chemiluminescence of the second complex or the displaced detectabletag and a combination thereof to obtain a measured observable. Themeasured observable is compared with a characterized observable, whereinthe characterized observable corresponds to the first complex.

The term “detectable tag” as used herein refers to a fluorescent orchemiluminescent tracer, which is conjugated to a donor product.Fluorescence is the preferred mode of detection for the invention. Asuitable detectable tag may be produced by conjugating for example, achemiluminescent tag or a fluorophore tag, to the donor product moleculein such a way that it does not interfere significantly with antibodybinding (i.e., most likely attached via the adenine portion of thenucleotide). Fluorophores applicable to the methods of the presentinvention include but are not limited to fluorescein, rhodamine, BODIPY™(DIPYrromethene BOron Difluoride), Texas Red, and derivative thereofknown in the art. Rhodamine conjugates and other red conjugates may besynthesized and optimized as detectable tags because their higherwavelength emission is less subject to interference fromautofluorescence than the green of fluorescein.

Chemiluminescent tags applicable to the invention include Lumigen TMA-6and Lumigen PS-3 (Lumigen, Inc., Southfield, Mich.) which have adequatechemiluminescence quantum yield. These reagents possess an easilymeasured signal by virtue of an efficient chemiluminescent reaction witha predictable time course of light emission. Furthermore,chemiluminescent tags contribute little or no native backgroundchemiluminescence. Also, measurement of light intensity is relativelysimple, requiring only a photomultiplier or photodiode and theassociated electronics to convert and record signals. In addition,chemiluminescent signals can be generated in an immunoassay using enzymefragment complementation methods, where the detectable tag would be thedonor product conjugated to fragment A of a reporter enzyme, and itsdisplacement from antibody by the donor product generated in the grouptransfer reaction would allow it to associate with fragment B of theenzyme resulting in formation of a catalytically active reporter enzymecapable of acting on a chemiluminescent substrate. This method has beendescribed using β-galactosidase as a reporter enzyme in U.S. Pat. No.4,708,929.

The term “immunoassay” as used herein may refer to a number of assaymethods wherein the product is detected by an antibody such as forexample a homogenous assay, homogeneous fluorescence immunoassay, ahomogeneous fluorescence intensity immunoassay, a homogeneousfluorescence lifetime immunoassay, a homogeneous fluorescencepolarization immunoassay (FPIA), a homogeneous fluorescence resonanceenergy transfer (FRET) immunoassay or a homogenous chemiluminescentimmunoassay, or a non-homogenous assay such as enzyme-linked immunoassay(ELISA) and a combination thereof.

The term “fluorescence polarization immunoassay” or “FPIA” as usedherein refers to an immunoassay for detecting the products of grouptransfer reactions. Fluorescence polarization (FP) is used to studymolecular interactions by monitoring changes in the apparent size offluorescently-labeled or inherently fluorescent molecules (Checovich, W.J., et al., Nature, 1995, 375:254-6; Owicki, J. C., J Biomol Screen,2000, 5:297-306). When a small fluorescent molecule (tracer) is excitedwith plane polarized light, the emitted light is largely depolarizedbecause the molecule rotates rapidly in solution during the fluorescenceevent (the time between excitation and emission). However, if the traceris bound to a much larger receptor, thereby increasing its effectivemolecular volume, its rotation is slowed sufficiently to emit light inthe same plane in which it was excited. The bound and free states of thefluorescent molecule each have an intrinsic polarization value, a highvalue for the bound state and a low value for the free state. In apopulation of molecules, the measured polarization is a weighted averageof the two values, thus providing a direct measure of the fraction ofthe tracer molecule that is bound. Polarization values are expressed asmillipolarization units (mP), with 500 mP being the maximum theoreticalvalue.

In practice, small molecules like fluorescein have polarization valuesof approximately 20 mP, and when bound by an antibody their polarizationincreases by 100 to 400 mP. In a competitive FPIA, a fluorescent tracermay be displaced from binding to antibody by the donor product, as shownin FIG. 1. The signal is proportional to the difference in the boundversus free tracer fractions, thus both the dynamic range and thesensitivity of the assay are dependent upon the affinity of the antibodyfor the tracer and the donor product. To establish a suitable dynamicrange for an FPIA, approximately 70-80% of the tracer must be bound toantibody in the absence of competitor.

For example, FIG. 1 shows a schematic of a competitive FPIA for the SULTreaction product PAP in which the PAP produced from the SULT reactioncompetes with the tracer (fluorescently tagged PAP), for binding toanti-PAP antibody. In this format, the starting polarization of thetracer is high because it is almost all bound to antibody, and itdecreases as the reaction proceeds and the tracer is displaced from theantibody. The amount of PAP produced in a SULT reaction can bequantified by using the following equation:

${\log\mspace{11mu}\left\lbrack {{PAP}\mspace{14mu}{product}} \right\rbrack} = {{\log\;\frac{\left( {{highest}\mspace{14mu}{polarization}\text{-}{mP}_{observed}} \right)}{\left( {{mP}_{observed}\text{-}{lowest}\mspace{14mu}{polarization}} \right)}} + {\log\mspace{14mu}{{IC}_{50}.}}}$

In the enzymatic assay encompassed by the present invention, the PAP forSULTs or ADP for kinases is produced in stoichiometric amounts with thesulfated product or phosphorylated peptide, respectively. Thus the useof a standard curve for PAP or ADP among other donor products will allowa direct measure of enzyme turnover.

The term “high throughput screening” or “HTS” as used herein refers tothe testing of many thousands of molecules (or test compounds) for theireffects on the function of a protein. In the case of group transferreaction enzymes many molecules may be tested for effects on theircatalytic activity. HTS methods are known in the art and they aregenerally performed in multiwell plates with automated liquid handlingand detection equipment; however it is envisioned that the methods ofthe invention may be practiced on a microarray or in a microfluidicsystem.

The term “library” or “drug library” as used herein refers to aplurality of chemical molecules (test compound), a plurality of nucleicacids, a plurality of peptides, or a plurality of proteins, and acombination thereof. Wherein the screening is performed by ahigh-throughput screening technique, wherein the technique utilizes amultiwell plate or a microfluidic system.

The terms “binding molecule” or “macromolecule” as used herein refers toan antibody or an inactivated enzyme.

The term “antibody” as used herein refers to a monoclonal, a polyclonalor recombinant antibody. The antibody is produced against adonor-product of a group transfer reaction and is able to preferentiallydistinguish between a donor-product and a donor in the presence of ahigh donor concentration. The antibody also exhibits specificity towardsat least one of a phosphate portion of a nucleotide, (i.e., an abilityto distinguish between a 5′-phosphate, a 5′-phosphosulfate, a5′-diphosphate and a 5′-triphosphate). For example, in the SULTreaction, the antibody of the present invention differentiates with highstringency between PAP and PAPS. The donor-product molecule for the SULTreaction, PAPS, differs only by the addition of a sulfate group linkedto the 5′ terminal phosphate. This may seem like a relatively smallstructural difference, but the demonstrated ability of antibodies todiscriminate between molecules that differ by a single phosphate—whichis very similar in size and structure to a sulfate group—provides animportant feature of the present invention. For example, an antibody maybe raised against the ribosyl phosphate portion of the molecule.Furthermore, the FPIA-based SULT assay method of the present inventionsuitably requires an antibody that specifically binds the reactionproduct PAP in the presence of excess PAPS.

In practicing the invention with respect to kinases, antibodies thatspecifically recognize ADP and not ATP may be generated in animals or byin vitro recombinant methods using ADP conjugated to a carrier proteinin such a way that the phospho-ribosyl portion of the molecule isexposed, but the adenine portion is largely hidden fromimmunoreactivity. In generating antibodies, it may be helpful to use anonhydrolyzable analog of ADP, such as one containing a methylene orsulfur group bridging the alpha and beta phosphates in order to preventhydrolysis of the immobilized hapten by nucleotidases or phosphatases inthe immunized animals. Alternatively, with respect to methyltransferasesand acetyltransferases, antibodies that specifically recognize smallmolecules other than nucleotides, respectively s-adenosyl homocysteineand Coenzyme A are used.

The term “inactivated enzyme” as used herein refers to a bindingmolecule that may bind to the donor product. In the case of a kinase,the ADP is the donor product and an inactivated nucleoside diphosphate(NDP) kinase may be the binding molecule. It is known that in manyenzymes, separate domains are involved in binding and in catalysis. Aselective destruction of the catalytic activity with preservation of thebinding properties by genetic engineering, allosteric inhibition, orother chemical means such as taking out cofactors, heme groups, etc. mayenable enzymes, particularly multi-subunit enzymes to function asspecific carriers for their substrates that are no longer able tochemically modify these substrates.

The term “antibody-detectable tag pair” as used herein refers to ananti-donor product antibody and detectable tag (fluorescent-donorproduct) molecule that allow detection of donor product produced in agroup transfer reaction. A suitable dissociation constant for binding ofthe antibody/detectable tag pair is 1×10⁻⁷M or lower, resulting in aminimal fluorescence polarization shift of 100 mP relative to theunbound detectable tag, and with minimal cross reactivity to the donormolecule at reaction concentrations. The optimal antibody-detectable tagpair may be identified by testing a number of different antibodiesgenerated using different sites of attachment to the donor molecule,different linkers to the carrier protein, and including somenon-hydrolyzable analogs for interaction with a set of detectable tagsgenerated by varying the chemistry of donor molecule attachment to afluorophore. The changes in fluorescence or chemiluminescence of thedetectable tag upon interaction with an antibody that could have afluorophore for example attached t it; may be used as a measure of theirinteraction.

The term “linker” as used herein refers to spacer arm structures. Thereare short linkers (i.e., carbamoyl and aminoethyl groups) that willsterically minimize the accessibility of the adenine ring and longer sixcarbon linkers that will allow more flexible presentation of theantigen. The linker molecule affects detectable tag characteristics in anumber of important ways that impact both its antigenic and fluorescenceproperties. There is generally a balance that must be struck betweenseparating the antigen from the fluorophore enough to allow unhinderedinteraction with antibody without creating too much freedom of motionfor the fluorophore. The former result in lowered affinity antibodybinding and in quenching of the fluorophore, whereas the latter reducesthe polarization shift upon antibody binding, thereby reducing thedynamic range of the assay. Different linkers can be interchanged usingrelatively simple chemistry, thus the linker is varied in a number ofways in efforts to optimize the antigenic and fluorescence properties ofthe detectable tag. Furthermore, approaches may be employed similar tothose described for PAP conjugation of an antibody involvingheterobifunctional linkers to first introduce spacer arms and/oraromatic substituents onto PAP, followed by reaction with reactivefluorescein derivatives. This approach greatly expands the range ofpossible linker structures.

Methods and Materials

The following experimental protocols of the invention are not limited tothe particular methodology, protocols, antibodies, enzymes, detectabletags, among other reagents described, as these may vary depending on thegroup transfer reaction. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention.

Development of Assay Reagents

In developing a successful HTS assay format for the invention theaffinity and specificity of assay reagent interaction, and the resultantchanges in detectable tag fluorescence properties ultimately define theoverall performance of the assay, including sensitivity, dynamic range,and signal to noise ratios. As such, to optimize this interaction,applicants have provided an iterative co-development approach as shownin FIG. 4 to most efficiently achieve detection, quantitation andscreening of group transfer reaction products.

In particular, FIG. 4 describes a strategy for iterative co-developmentof reagents for a SULT donor product FPIA: anti-PAP antibody andfluorescently labeled PAP detectable tag. FIG. 4 provides that toproduce diverse antigens for immunization of rabbits, PAP may beconjugated to carrier protein (BSA or KLH) via attachment through theC2, C8 and N6 amino group of adenine using linker molecules of varyinglengths as described below. The same PAP-linker-NH₂ intermediates usedfor antibody conjugation may be used to synthesize fluorescein-PAPconjugates to test as detectable tags. The resulting matrix ofantibodies and detectable tags may be tested for binding using FPassays, and the pairs that exhibit high affinity binding and maximal FPshifts may be used as the basis for further optimization of tracercharacteristics. Optimized Antibody-detectable tag pairs may be testedfor detection of PAP production in sulfation reactions with SULT1E1 oranother SULT isoform.

Also, it is envisioned that several PAP antigens and detectable tags maybe synthesized by conjugating the nucleotide to carrier protein andfluorescein, while retaining an overall structural bias that maximizesantibody recognition of ribosyl-phosphate moieties of the PAP moleculeand minimize crossreactivity with PAPS. Antibodies generated from thePAP antigens may be tested for interaction with the detectable tags toidentify the combinations that exhibit optimal binding and fluorescenceproperties of the novel assay encompassed by the present invention.

Generation of High Affinity Antibodies

In accordance with methods of the present invention, one skilled in theart may readily prepare a variety of specific antibodies (i.e.,polyclonal, monoclonal or recombinant) against antigenic nucleotidessuch as the ribosyl-phosphate portion of PAP or other chemical moleculessuch as Coenzyme A.

In general polyclonal antibodies against a range of PAP antigens aresuitably generated in rabbits. The injection of animals and collectionof serum may be performed according to the following protocol. Threerabbits may be immunized with each of the antigens that may bedeveloped. The yields of antiserum from a single rabbit (˜100 ml) aresuitable for many thousand to millions of FPIA assays depending on thetiter, affinity, and the multivalent nature of polyclonal-antigeninteractions resulting in very high affinity binding. As such, for thesereasons polyclonals are frequently used for FPIAs (Nasir, M. S. andJolley, M. E., Comb Chem High Throughput Screen, 1999, 2:177-90).

Also, encompassed within the scope of the present invention aremonoclonal antibodies which may be produced according to the methodsprovided in previously published protocols (Harlow, E. L., D., 1999),which are fully incorporated herein by reference. Also, recombinantsingle chain antibodies, may be generated using the in vitrocombinatorial evolution and display methods previously published(Schaffitzel, C., et al., J Immunol Methods, 1999, 231:119-35;Breitling, F., Dubel, S., 1999). Examples of the preparation ofrecombinant antibodies are described in U.S. Pat. Nos.: 5,693,780;5,658,570; 5,876,961 (fully incorporated by reference herein).

In order to help elicit an immune response, bovine serum albumin (BSA)and keyhole limpet hemocyanin (KLH) may be used as carrier proteins forconjugation of the PAP antigen. KLH antigens generally elicit a strongerimmune response in mammals, but also tend to be less soluble than BSAconjugates (Harlow, E. L., D., 1999). PAP may be attached to bothcarrier proteins via the C2, C8 and N6 amino group of adenine as shownin FIGS. 7-8. In accordance with the scope of the present invention,development of the FPIA-based SULT assay method requires an antibodythat specifically binds the reaction product PAP in the presence ofexcess PAPS. More suitably, the antibody is capable of recognizing theribosyl phosphate portion of the PAP molecule, and to differentiatebetween the 5′-phosphate of PAP and the 5′-phosphosulfate of PAPS.

Conjugation Methods

It is known in the art that conjugation to adenine will minimizeantibody cross reactivity with PAPS that could occur via binding to theadenine moiety (Goujon, L., et al., J Immunol Methods, 1998, 218:19-30;Horton, J. K., et al., J Immunol Methods, 1992, 155:31-40; Bredehorst,R., et al., Biochim Biophys Acta, 1981, 652:16-28), and the site ofhapten conjugation also can affect the affinity of the resultingantibodies (Crabbe, P., et al., J Agric Food Chem, 2000, 48:3633-8). Thehapten conjugation chemistries used are well described single or twostep synthetic approaches that have been used extensively forconjugating adenine nucleotides (Brodelius, P., et al., Eur J Biochem,1974, 47:81-9; Lindberg, M. and Mosbach, K., EurJ Biochem, 1975,53:481-6; Camaioni, E., et al., J Med Chem, 1998, 41:183-90). All of thestarting materials are commercially available, and it is understood thatthe reactions may proceed in good yield. These conjugation methods aredescribed below in further detail. In accordance with the methods of thepresent invention, it is envisioned that the present invention mayencompass but may not be limited by the following hapten conjugationchemistries.

Attachment to N6 Amino Group

The carbamoyl linkage: N6-carboxymethyl-PAP may be generated byalkylation of PAP with iodoacetic acid to form the 1-carboxymethylanalog, which rearranges in base to form the N6-carboxymethyl analog(Lindberg, M. and Mosbach, K., Eur J Biochem, 1975, 53:481-6). TheN6-carboxymethyl-PAP may be conjugated to carrier protein usingcarbodiimide coupling enhanced with N-hydroxysulfosuccinimide. A similarapproach could be used to generate high specificity 5′-AMP antibodies asdescribed in previously published protocols (Bredehorst, R., et al.,Biochim Biophys Acta, 1981, 652:16-28).

The C6 linkage: N6-aminohexyl-PAP (Sigma/Aldrich, St. Louis, Mo.) may bedirectly conjugated to carrier proteins by a) converting protein aminogroups (mostly lysines) to reactive thiols using N-succinimidylS-acetylthioacetate (Pierce, Rockford, Ill.) and b) conjugating theterminal amine group on the PAP linker to the protein thiols usingheterobifunctional amine-thiol reactive linkers (Pierce, Rockford,Ill.). It is noteworthy that the N6 amino group of adenosine is forpractical purposes unreactive; for instance it reacts very poorly withsuccinimidyl esters.

Attachment to Adenosine C2

The 2-chloroadenosine may be phosphorylated by reaction with phosphorousoxychloride followed by phosphorous trichloride to produce3′,5′-bisphospho-chloroadenosine. Subsequent reaction withethylenediamine and diaminohexane and purification by anion exchangechromatography may be used to produce the 2-aminoethyl- and6-aminohexyl-analogs of PAP at the 2 position of adenine (Brodelius, P.,et al., Eur J Biochem, 1974, 47:81-9). It is envisioned that thesemolecules may react with carrier proteins using heterobifunctionallinkers as described above.

Attachment to Adenosine C8

The C2 and C6 linkages to the 8 position of PAP may be generated usingthe same strategy described for the C2 position, except the startingmaterial may be 8-bromoadenosine (Sigma/Aldrich). Also, 8-azido-PAP(ICN, Costa Mesa, Calif.) may be directly bound to carrier proteins byUV irradiation.

Thin layer chromatography (TLC) may be used to monitor changes in theadenine ring absorption spectrum. Anion exchange (Dowex resin) and phaseseparations may be used for antigen purification. To assure high antigenpurity, the final PAP-linker molecules to be used for conjugation tocarrier proteins may be purified by HPLC and their identity verified bymass spectra and NMR analysis. The N6-carboxymethyl-PAP may beconjugated to methylated BSA, since use of the methylated carrierprotein has been reported to prevent carbodiimide reaction with theribose ring (Bredehorst, R., et al., Biochim Biophys Acta, 1981,652:16-28).

The other PAP analogs all tend to have a reactive amine at the end ofthe linker, which may be used for conjugation to BSA and KLH using morespecific amine and thiol chemistry, thereby avoiding reaction with theribose. The conversion of protein amines to thiols and linking viaheterobifunctional linkers may result in a higher density of antigenconjugation than direct conjugation to cysteines because there are manymore reactive lysines than cysteines in the carrier proteins. PAPdensity on the carrier protein may be determined by spectrophotometricanalysis for the adenine ring and conjugation chemistries may beadjusted if needed to obtain a high density (>10 PAP per proteinmolecule) and thus insure a robust immune response. It is envisionedthat about 8-10 different antigens may be synthesized in all, some withvery short linkers such as the carbamoyl and aminoethyl groups that willsterically minimize the accessibility of the adenine ring and also withlonger six carbon linkers that will allow more flexible presentation ofthe antigen.

Synthesis and Purification of Fluorescent PAP Molecules

In general, an FPIA detectable tag molecule can be divided into threedifferent structural components: the antigen, the fluorophore, and thelinker used to join them; an additional key structural variable is thesite of attachment of the linker to the antigen. It is understood by oneskilled in the art that suitable antigens, fluorophores and linkers arenot limited to what is specifically described herein. However, in asuitable aspect of this invention the fluorophore, fluorescein, may beused to conjugate it to the same sites on adenosine used for antigenconjugation to carrier protein. The same PAP-linker molecules that areused for conjugation to carrier proteins may be used for conjugation toseveral amine-reactive fluorescein derivatives. The use of a range ofreactive fluoresceins may introduce additional structural diversity inthe linker region. For instance, fluorescein succinimidyl esters areavailable with different length spacer arms, and DTAF(4,6-Dichlorotriazinylaminofluorescein) contains a bulky planarsubstituent adjacent to the site of attachment. Likewise, theintroduction of an aromatic ring to AMP near the site of carrier proteinconjugation (N6-benzyl AMP) enhances its affinity for antibody by40-fold relative to unmodified AMP (Bredehorst, R., et al., BiochimBiophys Acta, 1981, 652:16-28). Briefly, various PAP-linker moleculeswith amino termini may be reacted with amine-reactive rhodamine, TexasRed, and other red fluorophors and the resulting PAP-fluorophorsconjugates may be tested for antibody binding affinity and changes influorescence polarization.

Purification of PAP-conjugates can be performed by thin layerchromatography (TLC). TLC is used to separate reaction products becauseit allows several separations to be run in parallel, permitsidentification of fluorescent products by visualization and, providessufficient quantities of fluoresceinated conjugates for thousands ofbinding assays in a single chromatography run. Thus for pilot studies onfluorescent detectable tag molecules, TLC is superior to alternativeseparation methods like HPLC. Upon completion of the chromatography andelution of the PAP-fluorophore conjugates, the conjugates may beanalyzed for intensity, basal polarization (absence of antibody),stability, binding to anti-PAP antibodies, and competition by unlabeledPAP and PAPS, as described below.

Parameters Affecting the Antibody-detectable Tag Interaction

In order to create the most sensitive assay method for detecting,quantifying, and screening products of group transfer methods, theparameters affecting the affinity of the antibody-detectable taginteractions is carefully monitored. This is important, for example, inthe case of SULT1E1 where the potent substrate inhibition exhibited bysome acceptor substrates, may limit the amount of product that can begenerated. The affinity required for the novel SULT assay encompassed bythe present invention is estimated by considering the limiting case ofthe high affinity, inhibitory substrate, estradiol. In general, theK_(i) for estradiol is 80 nM and the K_(m) is about 20 nM (Zhang, H., etal., J Biol Chem, 1998, 273:10888-92), so using 50 nM estradiol in theassay and adjusting enzyme and reaction time so that 10% of thesubstrate is consumed—resulting in formation of 5 nM PAP—is a goodcompromise that may allow measurement of initial velocity withoutcausing significant inhibition.

To allow detection of 5 nM PAP the required affinity for antibodybinding to fluorescent-PAP (and unconjugated PAP) may be approximately25 nM. Most antibody-antigen interactions with either polyclonal ormonoclonal antibodies have a K_(d) between 1 nM and 1 μM (Harlow, E. L.,D., 1999). For instance, the anti-phosphotyrosine antibodies used inFPIA-based kinase assays, exhibit subnanomolar affinity forphosphorylated peptides and their fluorescent conjugates (Parker, G. J.,et al., J Biomol Screen, 2000, 5:77-88), and the anti-AMP polyclonalantibody described above bound to the nucleotide with an affinity of 100nM (Bredehorst, R., et al., Biochim Biophys Acta, 1981, 652:16-28). Itis difficult to predict how the structure of the antigen will affect theaffinity of the resulting antibodies, but the carrier protein and modeof conjugation both can have a profound effect (Crabbe, P., et al., JAgric Food Chem, 2000, 48:3633-8; Signorini, N., et al., Chem ResToxicol, 1998, 11:1169-75; Oda, M. and Azuma, T., Mol Immunol, 2000,37:1111-22). Accordingly, antibodies may be generated using PAPconjugated to two different proteins via different sites on PAP andusing different length linker regions.

Another factor affecting the sensitivity of PAP detection is poorsubstrates that are sulfated very slowly even at high concentrations. Inthese cases, the amount of SULT1E1 enzyme required becomes a limitingfactor because of cost considerations. Using a substrate that is turnedover at 1% the rate of the V_(max) with estradiol, it may require 30 ngof SULT1E1 to generate 5 nM PAP—a suitable detection limit in about 30minutes. This is well within the acceptable range, as it may mean that a100 mg batch of enzyme may support more than three million reactions.

Furthermore, the difference in polarization of the probe in the free andbound states defines the total “spread” or dynamic range for the assay.A change of less than 50 mP may be sufficient for semi-quantitativedetection of SULT1E1 activity, but a change of 100 mP or greater mayprovide both much greater flexibility in designing the assay format, andmore quantitative kinetic information. Accordingly, polarization isproportional to molecular volume, and the change in effective volumeupon binding of an antibody (150 kDa) to a small molecular weightfluorophore may be expected to cause an increase of at least 300 mP.However, there are other factors that can affect the observedpolarization of both the free and bound detectable tag, such as, thefluorophore is attached to PAP by a flexible spacer arm. Thus, theirmaximum polarization is a function of the extent to which antibodybinding decreases the mobility of the spacer and fluorophore; detectabletag “propeller effects” can lead to relatively low polarization valueseven for protein-bound fluorophores.

An additional consideration is whether there may be changes in theintensity of the fluorophores upon binding to receptor, includingquenching from amino acid functional groups with overlapping spectra.Polarization is independent of fluorescence intensity (Owicki, J. C., JBiomol Screen, 2000, 5:297-306); however, because it is a ratio ofintensity measurements taken in two planes, significant quenching mayaffect the antibody detectable tag interaction and result in decreasedassay sensitivity. The changes in the fluorescence properties of adetectable tag when it becomes bound by antibody are a function of bothinteracting molecules and are difficult to predict. Thus, it isenvisioned that different antibodies and detectable tag molecules willbe tested in a matrix fashion. Further optimization of the detectabletag structure or the assay buffer to limit mobility of the bounddetectable tag or to reduce quenching is also envisioned.

Identification of the Optimal Antibody/Detectable Tag Pair(s)

In order to identify optimal antibody/detectable tag pairs, a diverseset of fluorescein derivatives and linkage chemistries are generallyevaluated. The synthesis and purification methods described above resultin the isolation of several fluorescent products per reaction, so thatat least a hundred fluorescent conjugates may be evaluated, requiringthousands of individual FP assays. The homogenous nature of FP assaysand the availability of multiwell instruments makes the screeningefforts very rapid; the rate-limiting steps are usually synthesis andpurification, not testing for Antibody-detectable tag bindingproperties. The antibody/detectable tag testing may be carried out in aTecan Ultra multiwell FP reader in Tris or phosphate buffer in thepresence of a low concentration of carrier protein (0.01% Bovine GammaGlobulin) to prevent non-specific interactions. The initial screeninvolves looking for the following desirable properties: lowpolarization in the absence of antibody (<100 mP); high affinity bindingto the anti-PAP antibodies (K_(d)<100 nM); maximal difference inpolarization between the bound and free states (Δ mP >100 mP); minimalfluorescence quenching upon binding antibody; selectivity for PAP (i.e.,competitive displacement by PAP and not by PAPS or other adeninenucleotides); rapid association/dissociation kinetics; and lack ofinteraction with SULT1E1 or other assay components.

For initial antibody binding assays, each of the purified detectabletags may be quantified by measuring fluorescence intensity with theassumption that the intensity of the conjugated fluorescein is notquenched. This is not always the case, but those with significantquenching may be eliminated through the binding analysis because theyappear to have low binding affinity. Antibody/detectable tag pairs withpromising affinity (K_(d)<100 nM) and fluorescence properties may betested for the specificity of binding by competition with unlabeled PAPand PAPS and other nucleotides. Those Antibody/detectable tag pairs withthe most desirable FPIA properties may be examined in more detail toassess whether any changes in extrinsic conditions such as the additionof salt or detergent may improve their sensitivity or dynamic range.Additional detectable tags may be optionally synthesized and testedbased on the structure-activity relationships observed and thepredictions of computer-generated structural models.

APPLICABILITY OF THE INVENTION

The methods of the invention, as described in the examples below can beused as a measure of enzymatic activity in group transfer reactions. Themethods are of particular importance in the pharmaceutical industrysince they enable the analysis of group transfer enzymes in highthroughput screening laboratories, e.g. for identification of drugs thatcan act as enzymatic modulators, especially inhibitors, and fordetermining how potential drug molecules are metabolized.

There is an increasing number of group transfer enzymes that are beingtargeted for the development of new therapeutics. Kinases are currentlyof highest interest, but SULTs, UGTs, methyltransferases,acetyltransferases, and ADP-ribosyltransferases are also being targeted.As this list increases it will become impractical and extremelyexpensive to use specific tests for each enzyme, or for small subgroupsof enzymes that modify the same acceptor substrate. The assays and kitsof the present invention allow for the detection of all of the enzymesin a given group transfer enzyme family and provide a common end pointdetection system for all of them. This allows for greater ease of use,particularly in high throughput screening laboratories, where robots canbe set up with all the detection reagents and the various group transferenzymes and potential substrates or inhibitors of interest can be testedin a matrix fashion.

In addition, the involvement of group transfer reactions in theconjugative metabolism of drugs and other xenobiotics is an importantdeterminant in the level of their pharmacodynamics, pharmacokinetics andside effects. In this regard, the assays and kits of the presentinvention allow for the screening of potential drug molecules withSULTs, UGTs, methyltransferases and acetyltransferases in an HTS format.There are many different xenobiotic conjugating isoforms, and because ofthe generic nature of the method, the kits of the present inventionwould greatly simplify their testing in a matrix fashion.

Thus, the general methods of the invention may be applied to a varietyof different group transfer-related enzymatic processes, such as steroidhormone biosynthesis and function, xenobiotic metabolism, enzymereceptor regulation, and signal transduction in an effort to contributeto an integrated drug discovery approach discussed below.

Sulfation in Drug Discovery

Sulfation is a ubiquitous covalent modification used to regulate thelevels and activities of endogenous hormones and xenobiotics. In humansit is catalyzed by a family of eleven different sulfotransferase enzymes(SULTs), each with different, but overlapping substrate specificity andtissue distribution(Strott, C. A., Endocr Rev, 2002, 23:703-32).Endogenous substrates for sulfation include many important signalingmolecules, such as steroid hormones, catecholamines, and thyroidhormones; xenobiotics that are sulfated include drugs, promutagens andenvironmental endocrine disruptors (Strott, C. A., Endocr Rev, 2002,23:703-32; Glatt, H., et al., Mutat Res, 2001, 482:27-40; Coughtrie, M.W. and Johnston, L. E., Drug Metab Dispos, 2001, 29:522-8). Sulfation isreversible and can change the activity of signaling molecules, often byaltering their affinity for receptor proteins, thus it serves as anon-off switch for receptor ligands, much as phosphorylation serves thatrole for proteins. Moreover, the role of sulfation in xenobioticmetabolism is intertwined with its involvement in the regulation ofhormonal signaling and cell homeostasis. For instance, sulfationregulates the activity of endogenous ligands for specificneurotransmitter receptors, nuclear receptors, and protein kinases thatare drug targets for depression, breast cancer, and cardiovasculardisease (Plassart-Schiess, E. and Baulieu, E. E., Brain Res Brain ResRev, 2001, 37:133-40; Strott, C. A., Endocr Rev, 1996, 17:670-97;Kuroki, T., et al., Mutat Res, 2000, 462:189-95). Development of moreselective therapies for these disorders by pharmaceutical companies iscurrently hampered by a lack of molecular assays and purified SULTisoforms for high throughput screening (HTS) of potential SULTsubstrates and inhibitors.

There are two major classes of SULTs in humans that differ insolubility, size, subcellular distribution, and share less than 20%sequence homology. The membrane bound enzymes are located in the golgiapparatus and sulfonate large endogenous molecules such as heparan,glycosaminoglycans, and protein tyrosines (Strott, C. A., Endocr Rev,2002, 23:703-32). The cytosolic sulfotransferases, or SULTs sulfonatesmall molecular weight xenobiotics and hormones.

There are eleven known cytosolic SULT isoforms, which differ in theirtissue distribution and specificity. The nomenclature used is based onhomology at the amino acid level: SULTs within the same family,indicated by a number, share at least 45% amino acid identity, and thosewithin the same subfamily, designated by a letter, share at least 60%identity. The SULTs can be differentiated to some degree based onsubstrate specificity, though their substrate profiles overlapextensively, even between enzymes in the different families.

Structural studies have revealed that there is flexibility in thesubstrate binding sites and that ligand binding elicits the transitionto a more ordered structure, which may contribute to the broad substratespecificity of the SULTs (Bidwell, L. M., et al., J Mol Biol, 1999,293:521-30). For this reason, it is not likely that computationalapproaches such as molecular docking may be applicable for identifyingSULT substrates and inhibitors; this increases the value of the HTSassays encompassed by the present invention.

It is envisioned that the assays of the invention may be used in an HTSformat to provide for example, SULT metabolism information such aswhether the compound of interest interacts with one or more SULTisoforms, whether the compound is a substrate or inhibitor, and thekinetic parameters (IC₅₀, K_(m), V_(max)) for the interaction. The HTSassay encompassed by the present invention provides all of thisinformation by screening in two different modes: a) direct measurementof test compound turnover; and b) measurement of compound inhibition ofprobe substrate sulfation. In the direct turnover mode, compounds couldbe screened vs. a panel of SULT isoforms to profile compound sulfationby individual SULT isozymes. After isozyme identification, more detailedkinetic studies with the appropriate SULT isozyme could be used topredict in vivo clearance rates (Obach, R. S., et al., J Pharmacol ExpTher, 1997, 283:46-58), reducing the failure rate of compounds inclinical studies (Greco, G. N., E; Martin, Y C, 1998, 219-245). SULTinhibitors could be identified by using a known substrate—e.g. estradiolfor SULT1E1—and screening test compounds for inhibition. Isozymespecific kinetic and inhibition data will further improve drug discoveryby providing knowledge of metabolism by specific SULT alerting the drugdiscovery team to potential drug-drug interactions and pharmacogeneticissues. The contribution of genetic differences in SULTs to varyingresponses to therapeutics is an active area of investigation (Thomae, B.A., et al., Pharmacogenomics J, 2002, 2:48-56).

Identification of the SULT responsible for the metabolism of a drug willaid in judicious selection of the in vitro assays or animal models usedfor preclinical assessment of possible drug-drug interactions andtoxicology testing, thereby reducing inappropriate or unnecessaryexperimental animals use. Metabolism data can be used as a component ofrational drug design and lead optimization. A better understanding ofthe structure-activity relationships that define substrate specificityfor the various SULT isozymes may provide a basis for structuralmodifications of primary compounds to change their metabolism profile(Greco, G. N., E; Martin, Y C, 1998, 219-245). Accordingly, the methodsof the present invention will enable a better understanding of thestructure-activity relationships that define substrate specificity forthe various SULT isozymes.

Integrated Approach to Drug Discovery

The ability of the assays of the invention to be used as HTS assaysenables a rational, integrated approach to drug development fortherapeutic areas where for example, sulfation is a component of therelevant signal transduction biology. Some specific areas where the HTSmethods of the invention may be used include for example steroid hormonebased therapies. Sulfation, in accordance with the present invention,encompasses involvement in estrogen level regulation in mammary tumors,as well as androgen levels in prostate tumors. Furthermore, cortisolsulfation, though not well understood, inactivates the hormone forbinding to the glucocorticoid receptor. High throughput biochemicalassays for steroid ligand-receptor binding and the resultant binding oftranscriptional coregulator proteins are already commercially availableand being used by pharma HTS groups to find more selective steroidhormone modulators (Parker, G. J., et al., J Biomol Screen, 2000,5:77-88; Spencer, T. A., et al., J Med Chem, 2001, 44:886-97). As such,the availability of robust HTS assays for steroid sulfation may providean important addition to the arsenal of molecular tools available topharma groups focused on steroid signal transduction. For instance, theinhibition of SULT1E1 by compounds targeting the ER could causedeleterious effects by elevating estrogen levels in tumor cells.Molecules with SULT1E1 inhibitory properties could be culled or modifiedbased on studies using the assay methods of the present invention.

The modulation of neurosteroids is being investigated as a novelpharmacological approach to controlling neural excitatory balance(Malayev, A., et al., Br J Pharmacol, 2002, 135:901-9; Maurice, T., etal., Brain Res Brain Res Rev, 2001, 37:116-32; Park-Chung, M., et al.,Brain Res, 1999, 830:72-87). The methods encompassed by the presentinvention may suitably accelerate these efforts by allowing facilescreening of endogenous and synthetic neurosteroids forsulfoconjugation, offering insight into the fundamental biology as wellas providing a tool for lead molecule identification and optimization.The need for better molecular tools is accentuated by the fact thatthere is already a sizeable over the counter market for DHEA as an“anti-aging” dietary supplement purported to alleviate age relatedsenility and memory loss (Salek, F. S., et al., J Clin Pharmacol, 2002,42).

Furthermore, it is envisioned that the methods of the present inventionmay suitably identify drug targets with respect to cholesterol sulfatein the regulation of cholesterol efflux, platelet aggregation and skindevelopment in treatments for cardiovascular disease and perhaps someforms of skin cancer. In this instance, a sulfotransferase—most likelySULT2B1b—could become the drug target, and molecules that selectivelyinhibit this isoform may need to be identified. The availability of afull panel of the human SULTs and a robust HTS assay method of thepresent invention may be valuable to such an effort.

Glucuronidation in Drug Discovery

Drug metabolism problems such as production of toxic metabolites andunfavorable pharmacokinetics cause almost half of all drug candidatefailures during clinical trials (Obach, R. S., et al., J Pharmacol ExpTher, 1997, 283:46-58). All of the major pharmaceutical companies haverecognized the need to consider pharmacokinetic and pharmacogenomicconsequences early in the drug discovery process resulting in animmediate need for high throughput in vitro methods for assessing drugmetabolism. Aside from P450-dependent oxidation, glucuronidation isperhaps the most important route of hepatic drug metabolism. A broadspectrum of drugs are eliminated or activated by glucuronidationincluding non-steroidal anti-inflammatories, opioids, antihistamines,antipsychotics and antidepressants (Meech, R. and Mackenzie, P. I., ClinExp Pharmacol Physiol, 1997, 24:907-15; Radominska-Pandya, A., et al.,Drug Metab Rev, 1999, 31:817-99). Despite their importance, the broadand overlapping substrate specificity of the hepaticUDP-glucuronosyltransferases (UGTs) that catalyze glucuronidationremains poorly understood because of a lack of flexible in vitro assaymethods. The primary reasons for this is that the catalytic assays usedrequire separation of reactants from products, which involvessubstrate-specific chromatographic steps and thus is not practical in anHTS format.

Two UGT families (e.g., UGT1 and UGT2) have been identified in humans;although the members of these families are less than 50% identical inprimary amino acid sequence, they exhibit significant overlap insubstrate specificity. The members of the UGT1 family that are expressedin human liver, where the majority of xenobiotic metabolism takes place,include UGT 1A1, 1A3, 1A4, 1A6, and 1A9. Although the UGT2 family hasnot been studied as extensively, it is known that UGT2B4, 2B7, 2B10,2B11 and 2B15 are expressed in the liver. Mutations in UGTs are known tohave deleterious effects, including hyperbilirubinaemia which occurswith a frequency of 5-12% (Weber, W., 1997) and can lead toneurotoxicity and in severe cases, death. As is the case for other drugmetabolizing enzymes such as P450s, interindividual differences in UGTexpression levels have been observed and linked to differences in drugresponses (Iyer, L., et al., J Clin Invest, 1998, 101:847-54). Forinstance, low expression of UGT1A1, as in patients with Gilbert'ssyndrome, has been associated with the toxicity of Irinotecan, apromising anticancer agent (Wasserman, E., et al., Ann Oncol, 1997,8:1049-51). In addition, UGT upregulation in tumor tissues has beenidentified as a possible cause of anticancer drug resistance (Franklin,T. J., et al., Cancer Res, 1996,56:984-7; Takahashi, T., et al., Jpn JCancer Res, 1997, 88:1211-7).

All of the known UGTs exhibit broad substrate specificity, with a singleisozyme catalyzing glucuronidation of a broad range of structurallyunrelated compounds; not surprisingly there also is a great deal ofoverlap in the specificities of UGT isozymes (Radominska-Pandya, A., etal., Drug Metab Rev, 1999, 31:817-99). With regards to biotransformationof endogenous molecules, UGT 1A1 is clearly the predominant isoforminvolved in glucuronidation of the tetrapyrrole, bilirubin, resulting inits excretion. Beyond this, it is difficult to make generalizationsregarding specificity because of the lack of systematic studies withmost of the recently identified isoforms. Numerous endogenous steroidshave been identified as aglycones for most of the hepatic isoformsinclude including 1A1, 1A3, 1A4, 2B4, 2B7, and 2B15. Lipids and bileacids serve as substrates for 2B4 and 2B7, and recently retinoids havebeen identified as substrates for some isoforms from both families. Thestructural diversity of known xenobiotic aglycones is very broad; itincludes many drugs and drug like molecules including tertiary aminessuch as imipramine, non-steroidal anti-inflammatories (NSAIDs) such asacetominophen and naproxen, opioids such as morphine and codeine, andcarboxylic acid containing drugs such as clofibric acid.

In the short term, pharmaceutical companies have an immediate need forbetter methods to determine whether their potential drug candidates willbe glucuronidated in vivo, and if so by which UGT isoform. And in thelong term, developing the ability to predict the metabolism of drugs byglucuronidation will require a systematic effort to fully define the“chemical space” recognized by each of the key hepatic UGTs. Theproposed Phase II studies will generate the molecular tools required forthis effort, including HTS assay methods that can be used to rapidlyscreen large numbers of diverse chemicals for binding and metabolism byisolated UGT isoforms.

Pharmaceutical research and development is time consuming, expensive,and inefficient, resulting either in higher costs or lower availabilityof new therapies for the U.S. health care consumer. Currently,development of a new drug in the USA requires ten to fifteen years and atotal R&D outlay of $400 to $750 million. While clinical trials are themost expensive phase of development, typically accounting for 30-50% ofthe total R&D cost, only 10% of all drug candidates tested in clinicaltrials ultimately are commercialized (Obach, R. S., et al., J PharmacolExp Ther, 1997, 283:46-58). Moreover, an analysis of the reasons fordrug candidate attrition during clinical trials confirms that some ofthe key determinants of the success or failure of a compound are afunction of its metabolism, including rate of clearance(pharmacokinetics), potential for interference with the metabolism ofother drugs, and potential for generating toxic metabolites. Thecombination of poor pharmacokinetics—usually excessively rapidclearance—and toxicity cause over 50% of all clinical failures. Gaininga better understanding of how potential drug candidates are metabolizedearly in the discovery phase would improve the success rate inpreclinical and clinical studies resulting in more efficient drugdevelopment, and increased economy and availability of therapies.

It is envisioned that the immunoassay (i.e., FPIA-based donor productassay) for UGTs will be used in a manner very similar to that describedfor SULTs for determining whether potential drug candidates interactwith any of the known UGT isoforms. Using the method of the invention,it will be possible to determine whether compound of interest interactswith a one or more UGT isoforms, and if so, whether it is a substrate orinhibitor. Also one can identify the kinetic parameters (IC₅₀, K_(m),V_(max)) for the interaction between the compound of interest andenzymatic isoform. It should be noted that recombinant forms of the manyof the UGT isoforms are already available (Invitrogen,Becton-Dickinson). The information obtained with these HTS assays can beused in the following ways such as after isozyme identification, moredetailed kinetic studies with the appropriate UGT isozyme can be used topredict in vivo clearance rates, reducing the number of compounds thatfail in clinical studies due to poor pharmacokinetics. Also, theknowledge of metabolism by a specific UGT alerts the drug discovery teamto potential pharmacogenetic problems, since genetic differences in UGTlevels are recognized as an important factor in varying responses totherapeutics. Furthermore, the identification of the UGT responsible forthe metabolism of a drug will aid injudicious selection of the in vitroassays or animal models used for preclinical assessment of possibledrug-drug interactions and toxicology testing, thereby reducinginappropriate or unnecessary use of animals for experiments. Also,metabolism data can be used as a component of rational drug design. Abetter understanding of the structure-activity relationships that definesubstrate specificity for the various UGT isozymes would provide a basisfor structural modifications of primary compounds to change theirmetabolism profile. Also, the testing of glucuronidated compounds canlead to the discovery of valuable prodrugs that are inactive untilmetabolized in the body into an active form.

Protein Kinases in Drug Discovery

There are more than 400 distinct kinases encoded in the human genome;elucidating their role in disease and identifying selective inhibitorsis a major pharma initiative. Kinase malfunction has been linked to allof the most important therapeutic areas, including cancer,cardiovascular diseases, inflammation, neurodegenerative diseases, andmetabolic disorders. Moreover, clinical validation of kinases as drugtargets has recently been shown in the cases of Herceptin and Gleevec,which inhibit aberrant tyrosine kinases that contribute to breast cancerand leukemia, respectively. High throughput screening (HTS)—the paralleltesting of many thousands of compounds for interaction with a drugtarget—has become the dominant mode of drug discovery. The total marketfor HTS assay reagents in 2002 was $474M, and approximately 20% ofscreening was done on protein kinases. Despite the high level ofinterest, pharma efforts to incorporate kinases into HTS programs arehampered by shortcomings with the assay methods. The most commonly usedHTS kinase assays rely on fluorescence-based immunodetection of aphosphorylated peptide reaction product, which varies with the substratespecificity of individual kinases. Time consuming reagent development isthus required for each kinase, or group of related kinases, andcomparison of results between assays is problematic. To overcome thistechnical hurdle the invention provides for a FPIA for detection ofadenosine diphosphate (ADP), a product of all kinase reactions. Thisassay will accelerate efforts to define kinase substrate specificity andto identify novel inhibitors by providing a universal catalytic assaythat can be used with any kinase and any acceptor substrate.

Protein Kinases are a large, diverse family with a key role in signaltransduction. Protein kinases, which catalyze the transfer of theterminal phosphate group from ATP or GTP to serine, threonine ortyrosine residues of acceptor proteins, are one of the largest proteinfamilies in the human genome. In the broadest senses, they can bedivided into serine/threonine or tyrosine kinases and soluble enzymes ortransmembrane receptors. In the most recent and comprehensive genomicanalysis, 428 human kinases were identified that comprise eightdifferent homology groups, which also reflect differences in substratespecificity, structure/localization and/or mode of regulation (Hanks, S.K., Genome Biol, 2003, 4:111). For instance, there are 84 members of theTyrosine Kinase group, which includes both transmembrane growth factorreceptors such as EGFR and PDGFR and soluble enzymes such as the Srckinases, 61 members of the cyclic nucleotide dependent group, ser/thrkinases which includes the lipid dependent kinases—the PKC isoforms, and45 members of the “STE” group, which includes the components of themitogenic MAP kinase signaling pathway.

Kinases are ubiquitous regulators of intracellular signal transductionpathways, and as such have come under intense focus by pharmaceuticalcompanies searching for more selective therapies for a broad range ofdiseases and disorders; they are second only to G-protein coupledreceptors in terms of pharma prioritization (Cohen, P., Nat Rev DrugDiscov, 2002, 1:309-15). Intracellular targets for phosphorylationinclude other kinases, transcription factors, structural proteins suchas actin and tubulin, enzymes involved in DNA replication andtranscription, and protein translation, and metabolic enzymes (Cohen,P., Trends Biochem Sci, 2000, 25:596-601). Phosphorylation can causechanges in protein catalytic activity, specificity, stability,localization and association with other biomolecules. Simultaneousphosphorylation at multiple sites on a protein, with differentfunctional consequences, is common and central to the integration ofsignaling pathways.

Diversity of Phosphorylation Sites.

Each kinase may phosphorylate one or more target proteins, sometimes atmultiple sites, as well as autophosphorylate within one or moreregulatory domains that control catalytic activity or interaction withother biomolecules. Defining the functional consequences of cellularphosphorylation profiles for normal and disease states is a majorproteomics initiative. However, to use this knowledge for deciding whichkinases to target for drug discovery, their specificity for acceptorsubstrates must also be delineated. Kinases recognize specific linearsequences of their target proteins that often occur at beta bends. Ingeneral, amino acids that flank the phosphorylated residue for three tofive residues on either side define a phosphorylation site. ThePhosphoBase database, which compiles known kinase phosphorylation sites,contains entries for 133 human kinases, less than a third of the totalkinases. Moreover, most, if not all of these specificity profiles areincomplete, as they only show one or two peptides that have beenidentified as substrates for each kinase. Though there is significantoverlap in substrate specificity among related kinases, there is noconsensus sequence that is phosphorylated by a large number of kinases.This situation complicates the incorporation of diverse or novel kinasesinto HTS assays that rely on detection of specific phosphorylatedproducts.

TABLE 1 Target Structure: Company (Phase) EGFR Small molecule:GlaxoSmithKline (I), Pfizer (II), WyethAyerst (I), AstraZeneca (III),OSI Pharmaceuticals Monoclonal: Imclone (III), Abgenix (II), Merck (I),Medarex/Merck (I) Her2/Neu Monoclonal: Genentech (Herceptin), Medarex/Novartis (I), Genentech (I) PDGFR/ Small molecule: Novartis (Gleevec)cKit/BCR- Abl Raf Small Molecule: Merck, Onyx/Bayer (II) Antisense: IsisPharmaceuticals (II) MEK Small molecule: Pfizer (II), Promega (I) CDKSmall molecule: Aventis (II), Cyclacel (I), Bristol-Myers Squibb (I) PKCSmall molecule: Novartis (II), GPC Bioteck (II), Eli Lilly (I)Antisense: Isis (III) Table 1. Selected clinical trials for developmentof kinase inhibitors as anticancer agents (Dancey, J. and Sausville, E.A., Nat Rev Drug Discov, 2003, 2: 296-313). Bolded drugs are approvedProtein Kinases in Cancer

The biological rationale for targeting kinases to intervene in cancer isfar too extensive to attempt an overview here. However, one of thedominant themes is the involvement of numerous kinases in controllingthe delicate balance between the rate of cell division (cell cycleprogression), cell growth (mass), and programmed cell death (apoptosis)that is perturbed in all cancers. Growth factor receptor tyrosinekinases (RTKs) are membrane-spanning proteins that transduce peptidegrowth factor signals from outside the cell to intracellular pathwaysthat lead to activation of progrowth and anti-apoptotic genes. Themajority of the fifty-eight RTKs in humans are dominant oncogenes,meaning that aberrant activation or overexpression causes a malignantcell phenotype. Not surprisingly, tyrosine kinases are beingaggressively pursued as anticancer drug targets and both small moleculeand monoclonal antibody inhibitors—Gleevec and Herceptin,respectively—have been clinically approved. Downstream signaling fromgrowth factor receptors occurs through multiple pathways involving bothser/thr and tyrosine kinases. One of the dominant kinases is the mitogenactivated protein kinase (MAPK) pathway, which includes Raf and MEKkinases; inhibitors of all of these kinases are currently being testedin clinical trials (Table 1) (Dancey, J. and Sausville, E. A., Nat RevDrug Discov, 2003, 2:296-313). Soluble tyrosine kinases, especially the11 oncogenes that comprise the Src family, also transduce mitogenicsignals initiated by RTKs and are being targeted by pharma (Warmuth, M.,et al., Curr Pharm Des, 2003, 9:2043-59). Following mitogenic signalsthrough RTKs that initiate entry into the G1 phase, progression throughthe cell cycle is regulated by sequential activation of phase-specifickinases in association with cyclin proteins. The cyclin dependentkinases represent yet another important group of kinases that pharma ispursuing in the hopes of inhibiting malignant cell proliferation(Table 1) (Elsayed, Y. A. and Sausville, E. A., Oncologist, 2001,6:517-37).

Kinases as Targets in Other Diseases

Pharma interest in kinases is most intensely focused on cancer, butextends to all of the key therapeutic areas. Table 2 shows recentreviews describing the biological rationale for pursuing kinases targetsfor a broad range of disorders; note that the targets overlap thosebeing pursued for cancer. Entire companies have formed on the basis oftargeting kinases for drug discovery. These include Sugen(http://www.sugen.com, now owned by Pfizer), Signase(http://www.signase.com/index.htm), and ProQinase(http://www.proginase.com/index.html).

TABLE 2 Disease Targets Inflammatory diseases, MAPK (Adams, J. L., etal., Prog Med Chem, 2001, 38: 1-60); arthritis MEK 1, 2 (English, J. M.and Cobb, M. H., Trends Pharmacol Sci, 2002, 23: 40-5) Type II diabetesand GSK-3 (Eldar-Finkelman, H., Trends Mol Med, 2002, 8: 126-32), PI-complications 3 Kinase (Jiang, G. and Zhang, B. B., Front Biosci, 2002,7: d903-7), PKCβ (Frank, R. N., Am J Ophthalmol, 2002, 133: 693-8), IRTK(Laborde, E. and Manchem, V. P., Curr Med Chem, 2002, 9: 2231-42), PKA(Musi, N. and Goodyear, L. J., Curr Drug Targets Immune Endocr MetabolDisord, 2002, 2: 119-27) Hypertension, PKC, var. isoforms (Malhotra, A.,et al., Mol Cell Biochem, 2001, cardiovascular 225: 97-107), Rho Kinase(Chitaley, K., et al., Curr Hypertens Rep, 2001, 3: 139-44) Neuropathy -CDK5 (Lau, L. F., et al., J Mol Neurosci, 2002, 19: 267-73); MAPKAlzheimer's disease, (Dalrymple, S. A., J Mol Neurosci, 2002, 19:295-9), CKDs (O'Hare, M., stroke et al., Pharmacol Ther, 2002, 93:135-43) Neuropsychiatric PKC α, PKC ε (Chen, G., et al., Bipolar Disord,2000, 2: 217-36) disorders Antimicrobials Histidine Kinases (Matsushita,M. and Janda, K. D., Bioorg Med Chem, 2002, 10: 855-67)Table 2. Listing of Links between Protein Kinases and Various Diseases.

It is envisioned that the FPIA-based donor product assay will be used toscreen drug libraries for inhibitors or activators of protein kinases.It will also be useful for screening peptides or proteins as acceptorsubstrates for kinases. In these applications, it will have thesignificant advantages over other methods such as the universal natureof the assay, simplified homogenous assay, no radioactivity, and theability to quantify enzyme turnover.

Universal Assay Method

This method will accelerate the incorporation of protein kinases intoHTS screening programs because it is truly generic: a single set ofdetection reagents can be used for all kinases and all acceptorsubstrates. An important advantage over most of the current assaymethods is the capability to use physiological protein acceptorsubstrates as well as short peptides.

Homogenous

This assay is a single addition, mix and read format. This is animportant factor driving decisions on assay selection in an automatedhigh throughput environment (High Tech Business Decisions, M., CA,Commisioned Market Analysis, 2002). In addition, if antibodies withsuitable binding kinetics are isolated, it allows a continuous assayformat that provides more detailed kinetic information than a stop-timeassay.

Fluorescence Detection

By employing fluorescent probes, the FPIA format eliminates radiationhandling, disposal and costs. It should be noted that over the last fewyears FP has become one of the key HTS assay platforms used by pharma(Owicki, J. C., J Biomol Screen, 2000, 5:297-306). It is expected thatin 2003 it will be used by pharma in approximately 12% of total primaryscreening assays; this is a doubling from the level of FP usage in 2001(High Tech Business Decisions, M., CA, Commisioned Market Analysis,2002). FP is a standard mode on several commercial HTS plate readers.

Quantitative

In the proposed enzymatic assay, the ADP is produced in stoichiometricamounts with the phosphorylated peptide or protein, thus the use of astandard curve for ADP will allow a direct measure of enzyme turnover.Though the use of FP for HTS applications is a relatively recentdevelopment, the use of FPIAs for quantitative detection of hormones andmetabolites in a diagnostic setting is very well established (Nasir, M.S. and Jolley, M. E., Comb Chem High Throughput Screen, 1999, 2:177-90).

EXAMPLES Example 1 Uridine Glucuronide Transfersase Assay

One embodiment of this application involves detecting the “donorproduct” of the UGT reaction using a competitive fluorescencepolarization immunoassay where the antibody-bound tracer has a highpolarization value which decreases when it is displaced by an analyte,such as UDP (as shown in FIG. 2). The main reagent required for thisassay is the production of an antibody that binds UDP with highselectivity and has negligible binding to the donor, UDP-glucuronic acid(UDPGA). This highly selective antibody is used in combination with acommercially available fluorescent UTP compound to establish the assay.

The polyclonal antibody produced against UDP required covalent bindingto a carrier protein. UTP was used as the hapten only because reactivederivatives of the triphosphate, but not the diphosphate, were readilyavailable that could be used for conjugation. It was reasoned that themajority of the triphosphate may be hydrolyzed to di- and mono-phosphatein animals. Several different chemistries for linking the UTP to carrierprotein were investigated, because the nature of the linkage can have aprofound affect on the resulting antibody specificity and affinity forantigen. Care was taken so that the linker molecule was attached to theuridine ring rather than the ribose or phosphate, thus maximizing theimmunoreactivity with the portion of the UDP molecule that maydifferentiate it from the donor, UDPGA.

Rabbit antiserum raised against a mixture of UTP and UDP conjugated toBSA and a commercially available tracer molecule, a fluorescentlylabeled UTP compound (ALEXA™-UTP, Molecular Probes) was added to wellsof a black multiwell plate (Thermo Labsystems Pt#7605) containing theindicated amounts of uridine nucleotides. ALEXA™-UTP was used as atracer for the FPIA experiments. Fluorescence polarization was read in aTecan Ultra plate reader after several hours of equilibration. Reactionconditions were as follows: 50 mM KPO₄ pH 7.4, 150 mM NaCl, 0.1 mg/mlBGG, 1 nM ChromaTide ALEX™ Fluor 488-5-UTP, 1.25 ul rabbit sera, 1001total volume.

The experimental results from the UGT reaction are provided in FIGS.8A-B. FIG. 8 shows titrations of antibody-tracer complex with variousuridine nucleotides using the first polyclonal antibody raised againstUDP/UTP and a commercially available tracer molecule (ALEXA™-UTP). It isnoted that the two graphs differ in the scale of the X-axis and thatcompetition by UDP, the donor product is half maximal at approximately10ÿM, whereas for UDPGA, the donor, half maximal displacement is higherthan 1 mM which is at least a 100× difference in selectivity.

Most relevantly, whereas UDP displaces the tracer at low micromolarconcentration, there is no detectable crossreactivity with UDPGA atconcentrations in excess of 100 μM. The crossreactivity with UTP is notproblematic for the proposed assay because it is not present, nor is itproduced, in UGT enzyme reactions. The ability of UDP to displace thetracer at low micromolar concentrations means that this antibody issuitable for detection of UDP produced in UGT enzyme reactions.Furthermore, since, through preliminary binding kinetic studies therehave been indications that the displacement of tracer by UDP is veryfast, use of antibody for monitoring UGT turnover in real time; i.e., acontinuous assay, is encompassed within the invention.

Despite their importance, the broad and overlapping substratespecificity of the hepatic UDP UGTs that catalyze glucuronidationremains poorly understood because of a lack of flexible in vitro assaymethods. The primary problem is that the catalytic assays used requireseparation of reactants from products, which involves substrate-specificchromatographic steps and thus is not practical in an HTS format. Byestablishing the concept for measuring the UGT reaction product, UDP ina fluorescent, homogenous format, the applicants have provided thetechnical foundation for solving this problem. Because the assaymeasures a product common to all UGT reactions, it allows a single modeof detection with any UGT isoform and any substrate. In addition, themethod does not require separation of reactants from products, and is asignificant improvement over other related assays because it usesfluorescence detection rather than colorimetric, making it moresensitive and more desirable for pharma HTS platforms, which have becomelargely reliant on fluorescence based detection. Also, it is acontinuous assay method, thus can provide real time kinetic data on UGTenzyme turnover. In addition the antibody-antigen binding reaction isless susceptible to interference from test samples than a conventionalcoupled enzyme reaction that has been used in the past for donor productdetection (Mulder, G. J. and A. B. D. Van Doorn, Biochem J., 1975, 151:p. 131-40). Thus, the novel assay method will enable screening ofdiverse compounds for metabolism by a panel of isolated UGT isozymes,which will greatly enhance preclinical metabolism studies, andpotentially reduce the clinical attrition rate.

Furthermore, the properties of the antibody produced by the applicantshave broader implications for development of HTS assays for otherimportant classes of enzymes. As shown in FIG. 8, UDP, but not UMPdisplaces the tracer, illustrating that the antibody is capable ofdifferentiating between nucleotides on the basis of a single phosphategroup. Thus, it is encompassed that antibodies against other “donorproducts” such as ADP for protein kinase reactions where the reactionproduct differs from the donor by a single phosphate may be produced.

Example 2 Kinase Assays

The assay method of the invention relies on FPIA detection of a kinasereaction product ADP that is produced in stoichiometric amounts withphosphorylated polypeptide. Similar to the UGT assay described above,the components of the FPIA based donor product kinase assay include anantibody to the donor product and a tracer comprised of a donor productconjugated with a detectable tag. The antibody is highly specific forADP (i.e., it is capable of recognizing ADP in the presence of excessATP and a fluorescent tracer). The antibody and tracer in a kinase assayare added to wells of a black multiwell plate at concentrations optimalfor the start of the assay. A suitable concentration of tracer is 1-2 nMand sufficient antibody is used to cause approximately 75% of themaximal polarization shift for the tracer. The acceptor substrate isadded to the wells at the desired concentration, generally 2-5-foldhigher than the K_(m) value. The acceptor substrate can be a peptide oran intact protein. One benefit of the invention is that any acceptorsubstrate can be used, whereas other kinase assay methods require aspecific acceptor for detection. A buffer that is compatible with thekinase to be assayed and the antibody-tracer interaction, generally aTris-Cl or phosphate buffer at about a neutral pH is used. With thebuffer, the other required components of the assay are added, includingATP at a concentration of 100 μM-5 mM, MgCl₂, and any other agentsrequired for activation or stabilization of the kinase. The kinaseenzyme is then added to initiate the reaction and the polarizationvalues are monitored in a multiwell reader such as the Tecan Ultra. Asthe reaction proceeds, ADP produced in stoichiometric amounts with thephosphorylated peptide or protein displaces the tracer from the antibodyresulting in decreased polarization values.

In screening for inhibitors, the compounds to be tested are generallydispensed into wells prior to addition of any other assay components,and control wells with no inhibitor added are included for comparison.

Each kinase may phosphorylate one or more target proteins, sometimes atmultiple sites, as well as autophosphorylate within one or moreregulatory domains that control catalytic activity or interaction withother biomolecules (Cohen, P., Trends Biochem Sci, 2000, 25:596-601).Defining the functional consequences of cellular phosphorylationprofiles for normal and disease states is a major proteomics initiative,and this knowledge can be used for deciding which kinases to target andtheir specificity for acceptor substrates in drug discovery. Thoughthere is significant overlap in substrate specificity among relatedkinases, there is no consensus sequence that is phosphorylated by alarge number of kinases. This situation complicates the incorporation ofdiverse or novel kinases into HTS assays that rely on detection ofspecific phosphorylated products.

Example 3 Sulfotransferase Assays

Expression and Purification of SULT1E1

In order to establish a sulfotransferase HTS assay method, SULT1E1, aSULT isoform, was first subcloned into an E. coli expression vector witha C-terminal 6× histidine tag and the expressed protein was purified byaffinity chromatography and characterized with respect to its physicaland enzymatic properties.

The purified protein migrated close to its calculated molecular weighton SDS-PAGE and more importantly, mass spectral analysis agreed veryclosely with predicted molecular weight (36,161 and 36,374 daltons,respectively). Because it was designed to retain a native N-terminus,the purified C-terminal 6×His construct was also subject to N-terminalsequencing; 15 amino acids were sequenced and they were identical withthe human SULT1E1 sequence in GenBank (NP005411). Further sequenceverification was obtained through mass spectral analysis and proteinsequencing.

To serve as a comparison, the enzymatic properties of the purifiedSULT1E1 were examined. Estradiol and estrone were used as physiologicalsubstrates and additional positive (α-naphthol) and negative (dopamine)control compounds tested to assess specificity. Two types of radioassayswere used for these studies, one using ³⁵S-PAPS (Foldes, A. and Meek, J.L., Biochim Biophys Acta, 1973, 327:365-74), and the other using³H-estradiol (Zhang, H., et al., J Biol Chem, 1998, 273:10888-92); bothhave been used extensively for SULT characterization and are describedbelow.

TABLE 4 Kinetic Parameters w/Estradiol Relative Reaction Rates withVarious acceptors V_(max) α- Construct K_(m) (nM) nmol/min/mgV_(max)/K_(m) Estradiol Estrone naphthol DHEA Dopamine hSULT1E1- 15 1308.67 100% 39% 41% 11% 2% 6xHis

Table 4 shows the enzymatic properties of SULT1E1 fusion proteins. K_(m)and V_(max) determinations were done using ³H-estradiol radioassays inwhich the radioactive sulfated product is separated from unreacted³H-estradiol by organic/aqueous phase extraction and counted in a liquidscintillation counter. Kinetic parameters were calculated from V vs. Scurves by nonlinear regression using GraphPad/Prizm software. Therelative reaction rates with various acceptors were determined using 400nM acceptor substrate in the ³⁵S-PAPS radioassay, in which the unreacted³⁵S-PAPS is precipitated as a barium-metal complex and the supernatantcontaining the ³⁵S-labeled sulfoconjugate is counted in a liquidscintillation counter. Basal reaction conditions in both cases were 10mM KPO₄ pH 6.5, 10 mM DTT, 1.5 mM MgCl₂, 10 mM PAPS, 0.5 ng SULT1E1,0.0025 to 15 mM acceptor.

The V_(max) and estradiol K_(m) values determined for the purifiedSULT1E compared favorably with published values for purified recombinantSULT1E1, which are 30-40 nmol/min/mg and 5-15 nM, respectively. Thepublished data on acceptor substrate specificity is more varied, but theresults reflect the general trend that estradiol and estrone are verygood substrates, α-naphthol is intermediate, and DHEA and dopamine arevery poor substrates (see Table 4). Thus all of the SULT1E1 constructsapplicants expressed showed native substrate specificity and catalyticrates similar to the highest published values.

Synthesis of Antigens and Generation of Antibodies

As to the actual development of the fluorescence-based HTS assay forsulfotransferases, there were four components showing a PAP FPIA thatdetected SULT catalysis: synthesis of antigens and generation ofantibodies; synthesis of tracer molecules; testing antibodies andtracers for interaction and specificity; and demonstrating detection ofSULT1E1 activity using optimal antibody-tracer pairs. Tracer synthesisefforts overlapped significantly with antigen synthesis efforts, as thesame reactive PAP derivatives were used for conjugating to carrierprotein and fluorophores.

Development of the proposed FPIA-based SULT assay method requires anantibody that specifically binds the product of the SULT reaction, PAP,in the presence of excess PAPS; i.e., an antibody that discriminates onthe basis of a single 5′-sulfate group. There is ample precedent forantibodies that discriminate between various nucleotides that differonly in the number of phosphates, which is similar in size and structureto a sulfate group as described above. However, there was no precedentfor generation of antibodies that specifically recognize PAP.

Small molecules like PAP must be conjugated to a carrier protein inorder to be used as an immunogen. Suitably, an antigen density of 10-20per carrier protein is optimal. As discussed above, the two elements ofour antigen synthesis strategy were a) synthesis and testing of severalantigens because the site of attachment to nucleotide and linkerstructure can profoundly affect the properties of the resultingantibodies (Crabbe, P., et al., J Agric Food Chem, 2000, 48:3633-8;Signorini, N., et al., Chem Res Toxicol, 1998, 11:1169-75; Oda, M. andAzuma, T., Mol Immunol, 2000, 37:1111-22), and b) conjugation via theadenine ring because this may allow free exposure of the 5-phosphategroup and limit immunoreactivity with the adenine portion of themolecule, resulting in antigens with the desired specificity. None ofthe nitrogens in the adenine ring were reactive enough to conjugatedirectly to protein or via crosslinking reagents, so reactive PAPderivatives were used as a starting point; the only commerciallyavailable reagent was N6-aminohexyl PAP. What initially appeared to bethe most straightforward approach—reacting N6-aminohexyl-PAP withcarrier proteins by carbodiimide coupling or amine-reactivecrosslinkers—generated unacceptably low antigen density, despite asignificant experimental effort.

To pursue alternative chemistries applicants outsourced the synthesis ofthe photoreactive molecules, 2- and 8-azido-PAP and C8-hexylamino-PAP(FIG. 6). FIG. 6 shows the synthesis of PAP antigens. 2- and8-azido-PAP, which were custom synthesized by ALT, Inc. (Lexington, Ky.)were irradiated (254 nm) in the presence of BSA; unreacted nucleotideswere removed by filtration and dialysis. Final antigen densities of 7-12PAP/BSA were obtained as determined by absorbance of the adenine ring(shifted to 270-280 nm). Rabbits (3 per antigen) were immunized byLampire Biologicals (Ottsville, Pa.). N6-aminohexyl PAP (Sigma) wasconjugated to KLH using glutaraldehyde and injected into three rabbitsby Biosynthesis Corp. (Lewisville, Tex.). Immunization schedules weresimilar for all antigens and included 3-4 injections over a 4-6 weekperiod.

Antigens from the two photoreactive PAP derivatives were produced andsent out for antibody production; generation of antigen fromN6-aminohexyl PAP using glutaraldehyde crosslinking to KLH andproduction of antibody was contracted out to a separate company (FIG.6). All three antibodies were reported by the contract producers to bindPAP as determined by ELISA (data not shown).

In addition, applicants synthesized the reactive derivativesN6-carboxymethyl PAP and 2′-O-succinyl-PAP (FIG. 7); antibodies fromthese derivatives have not yet been produced. A detailed description ofAb binding properties using competitive FPIA is provided below followingthe description of tracer synthesis.

Synthesis of PAP-fluor Tracers

An FPIA tracer molecule can be divided into three different structuralcomponents: the antigen, the fluor, and the linker used to join them; anadditional key structural variable is the site of attachment of thelinker to the antigen. Because identification of a tracer is largelyempirical, applicants used a variety of linkers to join PAP andfluorescein via different sites on each molecule; in most cases thefinal linker region is a composite of the reactive fluorescein and PAPmolecules used.

The antibody strategy was to conjugate through the adenine moiety inorder to generate antibodies that bind specifically to theribosyl-phosphate group of PAP. The same sites are the obvious sites forfluor conjugation as well in order to leave the desired immunoreactiveportion of the tracer molecule free to bind Ab. Also, fluorescein wasthe preferred fluor used for conjugation. Though red-shifted fluors suchas rhodamine are more desirable for HTS applications, development of FPtracers with fluorescein is usually more straightforward because it isless prone to non-specific binding effects and there are numerousactivated derivatives available. As to the linker molecule, it affectstracer characteristics in a number of important ways that impact bothits antigenic and fluorescence properties. There is generally a balancethat must be struck between separating the antigen from the fluor enoughto allow unhindered interaction with antibody without creating too muchfreedom of motion for the fluor. The former can result in loweredaffinity Ab binding and in quenching of the fluor, whereas the latterreduces the polarization shift upon Ab binding, thereby reducing thedynamic range of the assay.

N6-aminohexyl PAP was the only activated PAP molecule used for immunogensynthesis that was useful for tracer synthesis; the photoactivationreactions required to conjugate the two azido-PAP derivatives may beinefficient for joining two small molecules. To provide PAP moleculesactivated at different positions and with different linker regions,applicants outsourced the production of C8-aminohexyl-PAP andsynthesized in house two PAP derivatives with carboxy-terminal linkers:N6-carboxymethyl-PAP and 2′-O-succinyl-PAP. Though the latter compoundis linked through the ribose hydroxyl rather than the adenine, thisapproach has been used to generate highly specific antibodies andtracers for cAMP (Horton, J. K., et al., J Immunol Methods, 1992,155:31-40).

Below are brief descriptions of the synthesis and purification ofactivated PAP molecules and their conjugation to various activatedfluors; tracer synthesis components are summarized in FIGS. 7 and 8. Ingeneral, PAP molecules with amino-terminal linkers were conjugated tofluors activated with succinimidyl esters (or isocyanate in one case)and PAP molecules with carboxy-terminal linkers were reacted withfluorescein derivates containing free amino groups using carbodiimidecoupling.

FIG. 7 illustrates structures of components of Tracer Synthesis. FIG. 7from left to right provides PAP molecules with amino-terminal linkersattached at the C8 and N6 position, amine- and carboxy-reactivefluorescein derivatives, and PAP molecules with carboxy-terminal linkersat the N6 and 2′-OH. The fluorescent PAP conjugates were separated byTLC and tested for binding to anti-PAP antibodies. In accordance to theinvention, sixteen reactions were run containing different combinationsof PAP and fluorescein derivatives and each reaction yielded 1-4fluorescent products that could be resolved by TLC. In all, more than 40tracers have been purified and tested for binding to Ab. N6-aminohexylPAP (Sigma) and all of the reactive fluorescein derivatives (MolecularProbes) are commercially available. 2′-O-succinyl-PAP andN6-carboxymethyl-PAP were synthesized as described below; C8-aminohexylPAP was synthesized by Jena Biosciences (Jena, Germany). FIG. 8 providesrepresentative final tracer structures.

Preparation of N6-carboxymethyl PAP

To prepare N6-carboxymethyl PAP, 100 mg PAP was incubated with 0.3 gIodoacetic acid in 1.2 mL aqueous, adjusted to pH 6.5 with LiOH. Thereaction proceeded at 30° C. for 5-7 days, periodically adjusting the pHto 6.5. The resulting 1-carboxymethyl-PAP product was precipitated withethanol and reconstituted in distilled water and the pH was adjusted to8.5 with LiOH. This reaction was heated at 90° C. for 1.5 hours to yieldthe N6-carboxymethyl PAP. This product was purified on a Dowexl-X2(200-400 mesh) column equilibrated in 0.3 M LiCl, pH 2.75. A gradientwas applied over 10 column volumes using 0.5M LiCl, pH 2.0.N6-carboxymethyl PAP eluted off the column as pure product and wasconfirmed by mass spectral analysis, ˜20% yield.

Preparation of 2′-O-Succinyl-PAP

To prepare 2′-O-Succinyl-PAP, 10 mg (0.024 mmole) PAP and succinicanhydride (43 mg, 0.426 mmole) were dissolved in distilled H₂Ocontaining 10% triethylamine (v/v, 1 mL) and shaken for 1.5 hours withreaction progress monitored using reverse phase thin layerchromatography (RP-TLC). Upon completion, the reaction was lyophilizedtwice to ensure no residual triethylamine remained. The expectedsuccinate was column purified using Fast Flow Sepharose-Q resin(Pharmacia) equilibrated with 50 mL 250 mM NH₄OAc, and eluted with alinear gradient of 500 mM to 750 mM NH₄OAc (50 mL of each). Fractionscontaining the product were concentrated on a rotovap followed bylyophilization to furnish 8.4 mg or 67% yield as determined byabsorbance measurement. Stock solution was stored at −20° C. for futureuse.

Preparation of Fluorescein Conjugates with Amino-activated PAP

To prepare fluorescein conjugates with amino-activated PAP, 10 μL of a100 mM solution of N6- or C8-aminohexyl-PAP in distilled H₂O wascombined in a screw cap vial with a molar equivalent of a fluoresceinsuccinimidyl ester, the reaction was brought to a final volume of 200 μLusing anhydrous dimethyl sulfoxide (DMSO), and shaken on a vortexer for24 hours. Reaction progress was followed by RP-TLC using H₂O as thedeveloping media. Reaction products were purified using preparative TLCon normal phase silica gel using 1:1 EtOH/0.5 M NH₄OAc. Fluoresceinlabeled compounds—generally 3-5 produced in each reaction—werevisualized using UV light, scraped from the TLC plates, and extractedfrom the silica gel using 1:1 MeOH/0.5 M NH₄OAc. Individual fractionswere shaken on a vortex for 1 hour wrapped in aluminum foil andcentrifuged at 4000 RPM for 8 minutes. The supernatants were trituratedto a separate labeled vial and the extraction process repeated. Combinedsupernatants were standardized to 50 nM solution for future use andfrozen in amber microfuge tubes at −20° C.

Preparation of Fluorescein Conjugates with Carboxy-activated PAP

To prepare fluorescein conjugates with carboxy-activated PAP, 10 ml of a100 mM solution of N6-carboxymethyl-PAP or 2′-O-succinyl-PAP indistilled H₂O was combined with 50 equivalents of1-ethyl-3-(dimethylaminopropyl) carbodiimide (EDC) (10 μL of a 1Msolution in anhydrous. DMSO) followed by 75 equivalents ofN-hydroxysuccinimide (15 μL of a 1000 mM solution in anhydrous DMSO),the reaction was brought to an intermediate volume of 180 μL withanhydrous DMSO and shaken for 48 hours. One molar equivalent (20 ml) offluorescein derivative with a free primary amine was then added and thereaction was incubated with vortexing for 24 hr. Reaction progress wasfollowed by RP-TLC using H₂O as the developing media. Upon completion ofthe reaction, fluorescent products were purified by normal phase silicaTLC and eluted as described above, except that 9:1 acetonitrile/2mMNH₄OAc, pH 5.5 was used as the TLC solvent and 8:2 acetonitrile/2 mMNH₄OAc, pH 5.5 was used for elution.

A total of 16 unique combinations of activated PAP and fluoresceinmolecules were reacted and from these, more than 40 fluorescent productswere isolated and tested for binding to antibodies. This approach can beviewed as sort of a “poor man's combinatorial chemistry,” applicantshave found it to be very successful for development of FP tracers inother instances. It is noted that the homogenous nature of FP assays andthe availability of multiwell instruments makes the screening effortsrelatively rapid.

Characterization of Anti-PAP Antibodies and Tracers

Analysis of antibody/tracer interaction was done by fluorescencepolarization. To measure fluorescence polarization (described brieflyabove), the emission intensity is measured in both the vertical andhorizontal planes, which are parallel and perpendicular to thevertically polarized excitation light. The emission light definespolarization: P=(horizontal intensity−vertical intensity)/(horizontalintensity+vertical intensity) Polarization values are reported as mP,which is 1000 times the polarization value (30 mP=0.03 P). Fluoresceinhas a polarization of approximately 20 mP at 37° C. in phosphate bufferat pH 7.0. Mathematically, the limiting polarization for fluorescein is500 mP; practically, an anti-fluorescein antibody bound to fluoresceingives a polarization of ˜450 mP. The currently available multiwell FPreaders such as the Tecan Ultra can read polarization values with greatprecision; i.e., detection of 1 nM fluorescein in the free and the boundstate with less than <3 mP standard deviation.

In a competitive FPIA, a fluorescently labeled antigen, a tracer, isdisplaced from binding to antibody by the analyte. The signal isproportional to the difference in the bound versus free tracerfractions, thus both the dynamic range and the sensitivity of the assayare dependent upon the affinity of the antibody for the tracer and thecompeting sample molecule. To establish a suitable dynamic range for anFPIA, at least 70-80% of the tracer must be bound to antibody in theabsence of competitor. This normally is achieved by using the tracer ata concentration 2-10 fold below the K_(d) and the antibody at aconcentration 2-3-fold over the K_(d). The useful concentration range ofthe assay then ranges from several-fold below the K_(d) concentration toabout 20-fold over the K_(d) concentration. For example, if the K_(d)for the tracer/antibody complex is 10 nM, then the range of detectionfor the sample may be from about 2 nM to about 200 nM. If the K_(d) isten fold less (1 nM), the sensitivity of the assay also improves about10-fold.

Acceptable properties for anti-PAP antibody and tracer in terms of theassay parameters affected are defined as 1) dynamic range: low tracerpolarization in the absence of Ab (<100 mP) and maximal difference inpolarization between the bound and free states (Δ mP>100 mP); 2)sensitivity: high affinity binding of tracer to the anti-PAP antibodies(K_(d)<100 nM), displacement by free PAP with a similar IC₅₀, andminimal fluorescence quenching caused by binding; 3) signal/noise: highAb selectivity; i.e., competitive displacement of tracer by PAP and notby PAPS or other adenine nucleotides; also lack of tracer interactionwith SULT1E1 or other assay component; and 4) a continuous assay: rapidassociation/dissociation kinetics.

Results of Ab-Tracer Interaction Studies

Initial screens for Ab-tracer binding were done by adding severalconcentrations of each antibody to 1 nm tracer in multiwell plates andmonitoring for increases in tracer polarization. With all of theantibodies, purification of IgG fractions using immobilized Protein A orProtein G was required to remove non-specific binding components. All ofthe data shown is using antibodies purified on Protein G (Pierce Nab™Protein G Spin Chromatography Kit). Tracer/Ab combinations that showedan interaction in the initial screen were analyzed in more detail forcompetitive displacement by PAP and selectivity of PAP over PAPS. Atotal of nine purified antibodies (3 antigens ×3 rabbits) were testedfor binding with 44 different fluorescein-labeled PAP tracers. Allassays were done in black 96-well plates or black low-volume 384-wellplates, which gave essentially identical results, and polarizationvalues were read on a Tecan Ultra with a fluorescein filter set(excitation at 485 nm to emission at 535 nm) (Ex₄₈₅/Em₅₃₅).

The binding isotherms in FIG. 9 were generated using the threeantibodies and two tracers shown in Table 5 below, and arerepresentative of experiments used to screen antibodies and tracers forbinding.

TABLE 5 Representative antibodies and tracers Abbreviation DescriptionAb 1781 8-azido-PAP-BSA immunogen Ab 1810 2-azido-PAP-BSA immunogen Ab3642 N6-aminohexyl-PAP-KLH immunogen N6-PAP-F8 N6-aminohexyl-PAPconjugated to 6-carboxyfluorescein succinimidyl ester C8-PAP-F14C8-aminohexyl-PAP conjugated to 6-carboxyfluorescein succinimidyl ester

Also, FIG. 9, provides binding isotherms for anti-PAP antibodies andPAP-fluorescein tracers. Shown are the N6-PAP-F8 tracer binding to Abs1781 ◯, 1810 ⋄, and 3642 □, and C8-PAP-F14 tracer with the sameantibodies: 1781 ●, 1810 ♦, 3642 ▪. Antibodies were serially dilutedtwo-fold in 50 mM phosphate buffer (pH 7.4) containing 150 mM NaCl, 0.1mg/mL BGG, and 1 nM tracer in a total volume of 100 uL in a 96-wellplate or 12 ul in 384-well plates (results in the two plates areidentical) and polarization values were read on the Tecan Ultra afterone hour incubation at room temperature.

The binding properties of antibodies generated from the same immunogenin different animals were similar, but not identical. In thisexperiment, the 3642 antibody bound both tracers significantly tighterthan 1781, and 1810 did not bind either tracer even at the highestconcentration tested. The maximal polarization values for 3642 bindingto N6-PAP-F8 and C8-PAP-F14 were 140 and 300 mP, respectively; unboundtracer polarization was approximately 20 mP, so the shift observed ismore than adequate for use in an FPIA. To test whether binding isspecific, competitive displacement by PAP was assessed.

FIG. 10 shows PAP and PAPS competition curves for Ab 3642 and the sametwo tracers, PAP and PAPS. N6-PAP-6F8 tracer is represented by the opensymbols: ◯(PAP), □ (PAPS). C8-PAP-F14 tracer is represented with theclosed symbols: ● (PAP), ▪ (PAPS). PAP or PAPS was serially dilutedtwo-fold in black mutiwell plates containing 50 mM phosphate buffer (pH7.4), 150 mM NaCl, 0.1 mg/mL BGG, 1 nM C8-PAP-F14, and 1.5 uL purifiedAb 3642 in a total volume of 12 μl or 1 nM N6-PAP-F8 and 0.5 uL 3642 Abin a 20 uL volume. Polarization values were read after one hour at roomtemperature.

The IC₅₀ for PAP with the C8-PAP-F14 tracer is 300 nM, low enough toallow use of these reagents for monitoring SULT1E1 activity. Note that amuch higher concentration of PAP (and PAPS) is required to compete offthe tighter binding N6-PAP-F8 tracer. This may be because in this casethe tracer has the same linker group as the immunogen used to generateantibody, and a population of antibody is recognizing the linker, makingthe tracer more difficult to displace with free PAP. FIG. 10 also showsthat PAPS is less effective than PAP at displacing the tracers from Ab3642.

Similar results were observed with the 1781 Ab as shown in FIG. 11,which includes complete curves for PAP, PAPS and several similarmolecules in competition experiments with the C8-PAP-F14 tracer. FIG. 11shows competition curves with two anti-PAP antibody/tracer combinations.Competitors were serially diluted two-fold in a black 384-wellmicrotiter plate containing 50 mM phosphate buffer (pH 7.4), 150 mMNaCl, 0.1 mg/mL BGG, 1 nM Tracer C8-PAP-F14, and either 1.5 uL Ab 3642(A) or 3 uL Ab 1781 (B) in a total volume of 12 uL. After one hourincubation at room temperature, the polarization values were read on aTecan Ultra (Ex₄₈₅/Em₅₃₅). The mean and standard deviation of duplicatesfor all data sets are shown. The IC₅₀ values for PAP and PAPS with the3641/tracer and 1781/tracer combinations were 0.3 uM PAP, 3.8 uM PAPS;and 0.3 uM PAP, 1.5 uM PAPS, respectively.

The 1781 and 3642 antibodies exhibited a 5- and 13-fold selectivity forPAP over PAPS respectively, and much higher selectivity for PAP over allof the other nucleotides tested. The cross reaction with PAPS is higherthan expected given the lack of cross reaction with other adeninenucleotides. In this regard, it should be noted that most commercialPAPS preparations contain a significant fraction of PAP, but applicantspurchased HPLC-purified preparations that were analyzed at greater than95% purity, and took precautions in its storage and use to preventhydrolysis. In any event, these results clearly show that applicants cangenerate antibodies that bind selectively to PAP in the presence ofPAPS, which is a key feasibility issue for allowing sufficientsignal:noise and dynamic range in the proposed SULT assay. An additional10-fold increase in Ab selectivity for PAP over PAPS may be sufficientand is a very reasonable expectation using monoclonals rather thanpolyclonals.

The results observed with other antibodies and tracers tested weresimilar. The additional antibodies produced from theN6-aminohexyl-PAP-KLH and 8-azido-PAP-BSA immunogens bound many of thetracers and the Ab/tracer complexes could be displaced in all cases byunlabeled PAP, indicating that the interaction was specific. The 1810antibodies showed very poor or no tracer binding and were not testedfurther. The polarization of the free tracers ranged from 15-40 mP andincreases observed upon Ab binding ranged from less than 100 mP toalmost 300 mP. Interaction with some tracers was too weak to allowsaturation with Ab, so maximal polarization changes were not alwaysobserved. More than ⅔ of the tracers tested bound to both types of Ab,but none of the tracers synthesized from N6-carboxymethyl-PAP bound toantibodies generated from either immunogen.

The FPIA-based SULT Assay

Though applicants will need to produce an antibody that differentiatesbetween PAP and PAPS more effectively to develop a high quality assay,applicants were able to use the 3642 Ab and C8-PAP-F14 tracer to monitordetection of PAP produced in reactions containing the purifiedSULT1E1-cHis. In the initial experiment applicants sought to identifythe optimal PAPS concentration for maximal signal:noise. SULT1E1-cHiswas incubated with estradiol and varying concentrations of PAPS in thepresence of pre-formed Ab-tracer complex; SULT1E1 was present at a levelsufficient to rapidly drive the reactions to completion (FIG. 12).

FIG. 12 illustrates the effect of PAPS concentration on detection ofenzymatically generated PAP. The assay mixture included 200 ng ofSULT1E1-6×His (□) or assay buffer (▪) was added to wells containing 30mM phosphate (pH 7.4), 7 mM DTT, 8 mM MgCl₂, 75 mM NaCl, 0.5 mg/mL BGG,150 nM estradiol, 1 nM C8-PAP-F14 tracer, 12.5 uL Ab 3642, and varyingconcentrations of PAPS in a total assay volume of 100 μL. The plate wasincubated for 24 hour at 37° C. and read in a Tecan Ultra (Ex₄₈₅/Em₅₃₅).

As in a typical FPIA, the tracer was used at a concentration well belowthe K_(d) and the Ab was adjusted to a concentration that resulted inapproximately 85% of maximal tracer polarization in the absence ofcompetitor. FIG. 12 shows that there is a range of PAPSconcentrations—from approximately 1 to 5 mM—where the enzymaticallyproduced PAP causes a decrease in tracer polarization of approximately40 mP (the difference between the open and solid squares in FIG. 12).

That is, even though this antibody cross-reacts with PAPS significantly,it can be used in a competitive FPIA mode to detect PAP produced in aSULT reaction with saturating PAPS (Km for PAPS with SULT1E1 isapproximately 50 nM) with a dynamic range of 40 mP. Moreover, these werehomogenous reactions, or single addition reactions, in which all of thereaction and detection components were added at the start of thereaction, which is the preferred approach for an HTS assay. Similarresults are attained if the detection reagents are added after theenzymatic reaction is complete, thus the polarization signal is notaffected by the enzymatic reaction (data not shown).

Described above is an embodiment of how one of the Ab/tracer pairsapplicants produced was used for detection of PAP in SULT reactions thatare allowed to proceed to completion before reading. However, acontinuous assay is the most desirable format for HTS because it allowsaccurate enzyme rate determinations and precludes the need for a quenchstep. FIG. 13 shows that the 3642 Ab and C8-PAP-F14 tracer can be usedto continuously monitor SULT1E1 enzyme activity over time, allowingdetermination of enzyme rates with diverse substrates.

FIG. 13 provides graphs of continuous FPIA-based detection of SULTactivity with diverse substrates acceptor substrates (200 nM) were addedto wells containing 30 mM phosphate (pH 7.4), 7 mM DTT, 0.8 mM MgCl₂, 75mM NaCl, 0.5 mg/mL BGG, 2 uM PAPS, 200 ng SULT1E1-cHis, 1 nM C8-PAP-F14Tracer, and 12.5 uL Ab 3642 in a total volume of 100 uL. Controlreactions (top trace in each graph) lacked C-His-SULT1E1 and containedall other reaction components. The plate was incubated at room temp andpolarization read at 1 minute intervals.

The top trace in each graph is a control reaction lacking SULT1E1, thuspolarization does not change significantly, whereas in the reactionscontaining enzyme it decreases with time. The experimental setup wasvery similar to what might be done in an HTS setting: antibody andtracer were put into the SULT reaction mix, dispensed into a 384 wellplate containing different acceptor substrates, and the reactions werestarted by the addition of SULT1E1-cHis. The plates were then read atregular intervals and polarization values plotted as a function of time.In reactions with known SULT substrates, the polarization decreased overtime relative to control reactions lacking enzyme; as may be expected asenzymatically produced PAP displaces the tracer from Ab. Note that thereis no significant change in polarization when no acceptor substrate ispresent (graph f) or if SULT1E1 is absent (top trace in each graph),indicating that the PAPS molecule is sufficiently resistant tonon-productive chemical or enzymatic hydrolysis.

These results show that a generic, fluorescence-based activity assay forsulfotransferases is technically feasible. In these experiments, thecross reaction of the 3642 Ab with PAPS contributes significantbackground (i.e., decrease in polarization), limiting the dynamic rangeof the assay, but enzymatic rates can still be obtained from the linearportions of the velocity curves. The bar graph in FIG. 14 shows ratesfor each substrate calculated from linear portions of the velocitycurves in FIG. 13; though not a precise assay at this point, thisrank-ordering of substrates does correlate inversely with theirpublished K_(m) values and with the substrate profile that applicantsdetermined using purified SULT1E1 and the ³⁵S-PAPS radioassay (FIG. 14).Accordingly, FIG. 14 is a comparison of SULT1E1 acceptor substrateprofiles determined using the FPIA-based assay and the ³⁵S-PAPSradioassay. Rates of FPIA-based reactions were calculated from linearportions of curves shown in FIG. 13. It is noted that the ³⁵S radioassaydata is provided in Table 4. Acceptor substrates were used at 200 nM(FPIA) or 400 nM (³⁵S-radioassay).

Lastly, applicants used a known SULT inhibitor, DCNP, to show that theFPIA-based assay could be used to generate an inhibition curve (FIG.15). Specifically, FIG. 15 is a graph showing inhibition of SULT1E1 by2,6 Dichloro-4-nitrophenol (DCNP) measured with the FPIA-based assay.DCNP was serially diluted two-fold into wells in 46 uL of phosphateassay buffer (30 mM KPO₄ (pH 6.5), 0.5 mg/mL BGG, 15 mM DTT, 1.6 mMMgCl₂, 4 μM PAPS), followed by 50 μl of a 2X Antibody/tracer mix (5 uL3642 Ab/2 nM Tracer C8-PAP-F14), and 200 ng of SULT1E in a total volumeof 100 μl. The plate was incubated at room temp for 30 min, and read onthe Tecan Ultra. ΔmP values were calculated by subtracting the SULT1Ereactions from the no SULT1E controls. All values represent the mean ofreplicates.

The response of the FPIA assay to DCNP was validated by comparison withthe 35S-radioassay (data not shown); the K_(i) values determined withthe two assay methods were 7.8 mM and 11 mM, respectively. Thusapplicants have demonstrated that the assay can be used for detection ofsubstrates and inhibitors—both of the key intended HTS applications.

Taken together, these results clearly establish feasibility forproducing all of the components of the proposed FPIA based SULT assay:purified recombinant SULTs, antibody that selectively recognizes PAP inthe presence of PAPS, and fluorescent PAP tracers whose binding andcompetitive displacement from Ab can be detected by significant changesin polarization. In addition, applicants were able to demonstrate thatthe assay reagents could be used to detect SULT activity in a continuousmode, very similar to how the assay may be used in an HTS setting, thatthe response to different substrates is similar to the standardradioassay, and that the assay can be used for detection of aninhibitor.

Accordingly, it is envisioned that an antibody, suitably a monoclonalantibody with approximately 10-fold greater affinity and selectivity forPAP will be produced that will enable development of an assay withsuitable dynamic range and signal:noise for commercial HTS applications.

Example 4 Assay Systems

Although, the methods described herein may be utilized in a variety ofdifferent assay systems, in its simplest form, the present assay systemcomprises an assay receptacle in which the assayed reaction is carriedout, and a detector for detecting the results of that reaction. Inpreferred aspects, the assay receptacle is selected from a test tube, awell in a multiwell plate, or other similar reaction vessel. In suchcases, the various reagents are introduced into the receptacle andtypically assayed in the receptacle using an appropriate detectionsystem, described above such as a fluorescence polarization detector. Inaddition to a receptacle, a flat surface such as glass or plastic couldalso be used and the reaction components spotted onto the surface in adefined array (such as a microarray).

Alternatively, and equally preferred is where the reaction receptaclecomprises a fluidic channel, and preferably, a microfluidic channel. Asused herein, the term microfluidic refers to a channel or other conduitthat has at least one cross-sectional dimension in the range of fromabout 1 micron to about 500 micron. Examples of microfluidic devicesuseful for practicing the methods described herein include, e.g., thosedescribed in e.g., U.S. Pat. Nos. 5,942,443, 5,779,868, andInternational Patent Application No. WO 98/46438, the disclosures ofwhich are incorporated herein by reference.

In accordance with the above-described methods, it is envisioned that anenzyme mediated coupling reaction between a first and second reactantmay be carried out in channels of a microfluidic device. As such, byusing a microfluidics platform, it may be possible to mimic thecompartmentalization of a eukaryotic cell. This method could then beused to monitor the activity of group transfer reactions catalyzed byenzymes in a more native environment, in the context of other proteinsand with cellular components that may affect enzymatic activity.Therefore, data on the activity of enzymes that catalyze group transferreactions and the consequences of their inhibition can be obtained in asetting that will more accurately reflect an in vivo environment.

It is envisioned that these assay systems may be capable of screeningtest compounds that affect enzymatic reaction of interest. Optionally,devices used in accordance with the present invention are configured tooperate in a high-throughput screening format, e.g., as described inU.S. Pat. No. 5,942,443. In particular, instead of delivering potentialtest compounds to the reaction zone from a reservoir integrated into thebody of the device, such test compounds are introduced into the reactionzone via an external sampling pipettor or capillary that is attached tothe body of the device and fluidly coupled to the reaction zone. Suchpipettor systems are described in, e.g., U.S. Pat. No. 5,779,868 (fullyincorporated by reference). The sampling Pipettor is serially dippedinto different sources of test compounds which are separately andserially brought into the reaction zone to ascertain their affect, ifany, on the reaction of interest.

Movement of materials through the channels of these microfluidic channelnetworks is typically carried out using any of a variety of knowntechniques, including electrokinetic material movement (e.g., asdescribed in U.S. Pat. No., 5,858,195 (fully incorporated by reference),pressure based flow, axial flow, gravity flow, or hybrids of any ofthese.

Example 5 Assay Kit

Another embodiment of the invention is a kit for detecting andquantifying a Donor product of a group transfer reaction or a catalyticactivity generating the donor-product of a group transfer reaction. Thegeneral equation for the group transfer reaction includes adonor-X+acceptor→donor-product+acceptor-X, wherein the donor-product isdetected by the general detection reaction: firstcomplex+donor-product→second complex+displaced detectable tag. In itsmost simplest form, the kit for an FPIA immunoassay may include amacromolecule (i.e., antibody or an inactivated enzyme) and a tracer(displaced) and optionally the specific group transfer enzyme ofinterest. It is noted that the macromolecule and the tracer may eitherbe separate or incorporated into one solution vessel.

The kit may also include components such as, an activated donor, adetectable tag, acceptor substrates, inhibitors, buffers, cofactors,stabilizing agents, a set of instructions for using the kit, orpackaging and any combination thereof. In addition, the kit may beformatted for multiplex detection by using more than oneantibody/detectable tag pair where the detectable tags can bedifferentiated on the basis of the observables they produce. Theimmunoassay may be used to detect the donor product or the catalyticactivity generating the donor product.

In practicing the invention, it is encompassed that the kit may be usedfor screening a library for a molecule or a set of molecules, capable ofcontacting an enzyme, wherein the enzyme generates the donor-product ina group transfer reaction. The library may include at least one of aplurality of chemical molecules, a plurality of nucleic acids, aplurality of peptides, or a plurality of proteins, and a combinationthereof; wherein the screening is performed by a high-throughputscreening technique using a multi-well plate or a microfluidic system.

It is further envisioned that the macromolecule in the kit includes atleast one of an antibody, a polypeptide, a protein, a nucleic acidmolecule, an inactivated enzyme, and a combination thereof that iscapable of contacting the donor-product with high affinity. It isfurther envisioned that the kit optionally include at least one of asulfotransferase, a kinase or an UDP-glucuronosyl transferase, a methyltransferase, an acetyl transferase, a glutathione transferase, or anADP-ribosyltransferase and combination thereof.

Although FPIA is a suitable mode of detection, also encompassed withinthe scope of the invention are kits designed to be used for detectingdonor product or the catalytic activity generating the donor productthrough other means such as a homogenous assay, a homogeneousfluorescence intensity immunoassay, a homogeneous fluorescence lifetimeimmunoassay, a homogeneous fluorescence resonance energy transfer (FRET)immunoassay or a homogenous chemiluminescent immunoassay, or anon-homogenous assay such as enzyme-linked immunoassay (ELISA). In thecase of a homogeneous fluorescence intensity immunoassay or ahomogeneous fluorescence lifetime immunoassay, the kit could be composedof an antibody and fluorescent detectable tag where the intensity and/orlifetime of the detectable tag is different when it is bound to antibodyand than it is free in solution. The difference in fluorescence; i.e.,the assay signal, could be enhanced by modification of the antibody suchthat its interaction with the detectable tag results in a further changein its fluorescence properties; i.e., quenching, enhancement, or achange in the lifetime. In the case of a homogenous FRET immunoassay,the interaction of a first fluor associated with the detectable tag witha second fluor that is attached to the antibody—either directly or viaassociated binding molecules such as biotin and streptavidin—couldresult in the excitation of the second fluor (or the reverse), therebygenerating a fluorescence emission at a wavelength different from thatof the detectable tag. The second fluor could be a small organicmolecule or a luminescent lanthanide probe. (It is noted that lanthanideemission is not fluorescence and is referred to as luminescence-basedresonance energy transfer, or LRET). In the case of chemiluminescentdetection, the detectable tag could be the donor product bound to onefragment of an enzyme used for chemiluminescent detection such asβ-galactosidase. When the donor product-fragment-one complex isdisplaced from antibody by the donor product, it would then bind tofragment two of the enzyme, producing an intact, active enzyme thatwould be capable of producing a chemiluminescent signal with anappropriate substrate. In the case of an ELISA, the assay would not behomogenous, and would require donor product or antibody be immobilizedto the surface of multiwell plates. A secondary antibody conjugated to adetection enzyme would also be included in this format.

All publications cited herein are hereby incorporated by reference intheir entirety. In the case of conflict between the present disclosureand the incorporated publications, the present disclosure shouldcontrol.

While the present invention has now been described and exemplified withsome specificity, those skilled in the art will appreciate the variousmodifications, including variations, additions, and omissions that maybe made in what has been described. Accordingly, it is intended thatthese modifications also be encompassed by the present invention andthat the scope of the present invention be limited solely by thebroadest interpretation that lawfully can be accorded the appendedclaims.

1. A homogenous assay method for directly detecting a donor-productproduced in a group transfer reaction in the presence of a donormolecule, the method comprising the steps of: a) reacting a donormolecule which is adenosine triphosphate (ATP), with an acceptor in thepresence of a catalytically active enzyme to form the donor-productwhich is adenosine diphosphate (ADP) and an acceptor phosphate, suchthat the ATP is partially consumed; b) combining the ADP produced in agroup transfer reaction with a tracer and an antibody to provide areaction mixture, the antibody being specific for the ADP, the tracercomprising the ADP conjugated to a fluorophore, and capable of bindingto the antibody to produce a detectable change in fluorescencepolarization; c) measuring the fluorescence polarization of the mixtureto obtain a measured fluorescence polarization; and d) comparing themeasured fluorescence polarization with a characterized fluorescencepolarization value corresponding to a known ADP concentration todirectly detect the ADP produced in the group transfer reaction.
 2. Ahomogenous assay method for directly detecting a donor-product producedin a group transfer reaction, the method comprising: a) reacting a donormolecule which is an adenosine triphosphate (ATP) with a polypeptide, inthe presence of a kinase; b) forming the donor-product which is anadenosine diphosphate (ADP) and a phosphorylated polypeptide; c)contacting the ADP with a first complex comprising an antibody, thatspecifically recognizes the ADP and a tracer capable of producing anobservable; d) competitively displacing the tracer of the first complexby the ADP, to generate a second complex, ADP-antibody complex and adisplaced tracer, to directly detect the donor-product in the kinasereaction; and e) detecting a change in the observable produced by thetracer in the first complex bound to the antibody and the tracer.
 3. Ahomogenous assay method for directly detecting a donor-product producedin a group transfer reaction, the method comprising the steps of: a)providing a reaction mixture having products of the group transferreaction, a tracer and an antibody, wherein the reaction is a kinasereaction, wherein the products of the reaction include the donor-productwhich is an adenosine diphosphate (ADP), in the presence of a donormolecule which is an adenosine triphosnhate (ATP), wherein the antibodyis specific for the ADP, and wherein the tracer comprises the ADPconjugated to a fluorophore and is capable of binding to the antibody toproduce a detectable change in fluorescence polarization; b) measuringthe fluorescence polarization of the reaction mixture to obtain ameasured fluorescence polarization; and c) comparing the measuredfluorescence polarization with a characterized fluorescence polarizationvalue corresponding to a known ADP concentration to directly detect theADP produced in the kinase reaction.